Perhydrolase variant providing improved specific activity

ABSTRACT

An acetyl xylan esterase variant having perhydrolytic activity is provided for producing peroxycarboxylic acids from carboxylic acid esters and a source of peroxygen. More specifically, a  Thermotoga maritima  acetyl xylan esterase gene was modified using error-prone PCR and site-directed mutagenesis to create an enzyme catalyst characterized by an increase in specific activity. The variant acetyl xylan esterase may be used to produce peroxycarboxylic acids suitable for use in a variety of applications such as cleaning, disinfecting, sanitizing, bleaching, wood pulp processing, and paper pulp processing applications.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims benefit of U.S. Provisional Patent ApplicationNo. 61/425,569, filed Dec. 21, 2010, and U.S. Provisional PatentApplication No. 61/434,728, filed Jan. 20, 2011, each of which isincorporated by reference herein in its entirety.

TECHNICAL FIELD

The invention relates to the field of peroxycarboxylic acid biosynthesisand enzyme catalysis. More specifically, an enzyme catalyst comprising avariant enzyme having perhydrolytic activity is provided having anincrease in specific activity. Methods of using the present enzymecatalyst to produce peroxycarboxylic acids are also provided.

BACKGROUND

Peroxycarboxylic acid compositions can be effective antimicrobialagents. Methods of using peroxycarboxylic acids to clean, disinfect,and/or sanitize hard surfaces, textiles, meat products, living planttissues, and medical devices against undesirable microbial growth havebeen described (U.S. Pat. No. 6,545,047; U.S. Pat. No. 6,183,807; U.S.Pat. No. 6,518,307; U.S. Patent Application Publication No.2003-0026846; and U.S. Pat. No. 5,683,724). Peroxycarboxylic acids havealso been used in a various bleaching applications including, but notlimited to, wood pulp bleaching/delignification and laundry careapplications (European Patent 1040222B1; U.S. Pat. No. 5,552,018; U.S.Pat. No. 3,974,082; U.S. Pat. No. 5,296,161; and U.S. Pat. No.5,364,554). The desired efficacious concentration of peroxycarboxylicacid may vary according to the product application (for example, ca. 500ppm to 1000 ppm for medical instrument disinfection, ca. 30 ppm to 80ppm for laundry bleaching or disinfection applications) in 1 min to 5min reaction time at neutral pH.

Enzymes structurally classified as members of family 7 of thecarbohydrate esterases (CE-7) have been employed as perhydrolases tocatalyze the reaction of hydrogen peroxide (or alternative peroxidereagent) with alkyl esters of carboxylic acids in water at a basic toacidic pH range (from ca. pH 10 to ca. pH 5) to produce an efficaciousconcentration of a peroxycarboxylic acid for such applications asdisinfection (such as medical instruments, hard surfaces, textiles),bleaching (such as wood pulp or paper pulp processing/delignification,textile bleaching and laundry care applications), and other laundry careapplications such as destaining, deodorizing, and sanitization (U.S.Pat. Nos. 7,964,378; 7,951,566; and 7,723,083 and Published U.S. PatentApplication Nos. 2008-0176299, and 2010-0041752 to DiCosimo et al.). TheCE-7 enzymes have been found to have high specific activity forperhydrolysis of esters, particularly acetyl esters of alcohols, diolsand glycerols.

Published U.S. Patent Application No. 2010-0087529 to DiCosimo et al.,describes several variant CE-7 perhydrolases derived from severalThermotoga sp. having higher perhydrolytic specific activity and/orimproved selectivity for perhydrolysis when used to prepareperoxycarboxylic acid from carboxylic acid esters. Two of the variantsdescribed in Published U.S. Patent Application No. 2010-0087529,Thermotoga maritima C277S and Thermotoga maritima C277T, exhibited asignificant improvement in specific activity relative to the T. maritimawild-type enzyme.

Thermotoga maritima variants having higher peracid stability were alsoreported by DiCosimo et al. in U.S. Pat. Nos. 7,927,854; 7,923,233;7,932,072; and 7,910,347, and U.S. patent application Ser. No.12/632,462. Each variant was characterized as having an increasedperacetic acid formation to peracetic acid hydrolysis ratio (PAAF:PAAH)when compared to the T. maritima wild-type perhydrolase or the T.maritima C277S variant perhydrolase.

Several variants of the Thermotoga maritima C277S perhydrolase have beenidentified which have higher specific activity for the perhydrolysis ofesters when compared to the specific activity of the Thermotoga maritimaC277S (U.S. patent application Ser. Nos. 13/070,123; 13/070,130;13/070,135; 13/070,144; and 13/070,149 to DiCosimo et al.). However,there remains a need to identify additional variants having an increasein perhydrolytic specific activity.

The problem to be solved is to provide an enzyme catalyst comprising aCE-7 perhydrolase having higher specific activity for the perhydrolysisof esters when compared to the specific activity of the Thermotogamaritima C277S perhydrolase or the Thermotoga maritima C277Tperhydrolase.

SUMMARY

Nucleic acid molecules encoding the Thermotoga maritima acetyl xylanesterase variants C277S or C277T were mutated to create libraries ofvariant enzymes having perhydrolytic activity. Several perhydrolasevariants were identified having an increase in specific activity whencompared to the parent enzyme from which they were derived (i.e., theThermotoga maritima C277S perhydrolase or the Thermotoga maritima C277Tperhydrolase) under the same assay conditions.

In one embodiment, an isolated nucleic acid molecule encoding apolypeptide having perhydrolytic activity is provided selected from thegroup consisting of:

-   -   (a) a polynucleotide encoding a polypeptide having perhydrolytic        activity, said polypeptide comprising the amino acid sequence of        SEQ ID NO: 34;    -   (b) a polynucleotide comprising the nucleic acid sequence of SEQ        ID NO: 33; and    -   (c) a polynucleotide fully complementary to the polynucleotide        of (a) or (b).

In other embodiments, a vector, a recombinant DNA construct, and arecombinant host cell comprising the present polynucleotide are alsoprovided.

In another embodiment, a method for transforming a cell is providedcomprising transforming a cell with the above nucleic acid molecule.

In another embodiment, an isolated polypeptide having perhydrolysisactivity is provided comprising the amino acid sequence of SEQ ID NO:34.

In one embodiment, the variant polypeptide having perhydrolytic activityis characterized by an increase in specific activity (as determined byan increased amount of peroxycarboxylic acid produced) when compared tothe specific activity of the Thermotoga maritima C277S variant or theThermotoga maritima C277T variant (Published U.S. Patent Application No.2010-0087529 to DiCosimo at al.) under identical reaction conditions.

In another embodiment, a process for producing a peroxycarboxylic acidis also provided comprising:

-   -   (a) providing a set of reaction components comprising:        -   (1) at least one substrate selected from the group            consisting of:            -   (i) one or more esters having the structure

[X]_(m)R₅

-   -   -   -   -   wherein                -   X=an ester group of the formula R₆—C(O)O;                -   R₆═C1 to C7 linear, branched or cyclic hydrocarbyl                    moiety, optionally substituted with hydroxyl groups                    or C1 to C4 alkoxy groups, wherein R₆ optionally                    comprises one or more ether linkages for R₆═C2 to                    C7;                -   R₅=a C1 to C6 linear, branched, or cyclic                    hydrocarbyl moiety or a five-membered cyclic                    heteroaromatic moiety or six-membered cyclic                    aromatic or heteroaromatic moiety optionally                    substituted with hydroxyl groups; wherein each                    carbon atom in R₅ individually comprises no more                    than one hydroxyl group or no more than one ester or                    carboxylic acid group; wherein R₅ optionally                    comprises one or more ether linkages;                -   m=is an integer ranging from 1 to the number of                    carbon atoms in R₅; and wherein said esters have                    solubility in water of at least 5 ppm at 25° C.;

            -   (ii) one or more glycerides having the structure

-   -   -   -   -   wherein R₁═C1 to C21 straight chain or branched                    chain alkyl optionally substituted with an hydroxyl                    or a C1 to C4 alkoxy group and R₃ and R₄ are                    individually H or R₁C(O);

            -   (iii) one or more esters of the formula:

-   -   -   -   -   wherein R₁ is a C1 to C7 straight chain or branched                    chain alkyl optionally substituted with an hydroxyl                    or a C1 to C4 alkoxy group and R₂ is a C1 to C10                    straight chain or branched chain alkyl, alkenyl,                    alkynyl, aryl, alkylaryl, alkylheteroaryl,                    heteroaryl, (CH₂CH₂O)_(n), or (CH₂CH(CH₃)—O)_(n)H                    and n is 1 to 10;

            -   (iv) one or more acylated monosaccharides, acylated                disaccharides, or acylated polysaccharides; and

            -   (v) any combination of (i) through (iv);

        -   (2) a source of peroxygen; and

        -   (3) an enzyme catalyst comprising a polypeptide having            perhydrolytic activity, said polypeptide comprising the            amino acid sequence of SEQ ID NO: 34;

    -   (b) combining the set of reaction components under suitable        reaction conditions whereby peroxycarboxylic acid is produced;        and

    -   (c) optionally diluting the peroxycarboxylic acid produced in        step (b).

In another embodiment, a process is provided further comprising a step(d) wherein the peroxycarboxylic acid produced in step (b) or step (c)is contacted with a hard surface, an article of clothing or an inanimateobject whereby the hard surface, article of clothing or inanimate objectis disinfected, sanitized, bleached, destained, deodorized or anycombination thereof.

In another embodiment, a composition is provided comprising:

-   -   (a) a set of reaction components comprising:        -   (1) at least one substrate selected from the group            consisting of:            -   (i) one or more esters having the structure

[X]_(m)R₅

-   -   -   -   -   wherein                -   X=an ester group of the formula R₆—C(O)O;                -   R₆═C1 to C7 linear, branched or cyclic hydrocarbyl                    moiety, optionally substituted with hydroxyl groups                    or C1 to C4 alkoxy groups, wherein R₆ optionally                    comprises one or more ether linkages for R₆═C2 to                    C7;                -   R₅=a C1 to C6 linear, branched, or cyclic                    hydrocarbyl moiety or a five-membered cyclic                    heteroaromatic moiety or six-membered cyclic                    aromatic or heteroaromatic moiety optionally                    substituted with hydroxyl groups; wherein each                    carbon atom in R₅ individually comprises no more                    than one hydroxyl group or no more than one ester                    group or carboxylic acid group; wherein R₅                    optionally comprises one or more ether linkages;                -   m=is an integer ranging from 1 to the number of                    carbon atoms in R₅; and                -   wherein said esters have solubility in water of at                    least 5 ppm at 25° C.;

            -   (ii) one or more glycerides having the structure

-   -   -   -   -   wherein R₁═C1 to C21 straight chain or branched                    chain alkyl optionally substituted with an hydroxyl                    or a C1 to C4 alkoxy group and R₃ and R₄ are                    individually H or R₁C(O);

            -   (iii) one or more esters of the formula:

-   -   -   -   -   wherein R₁ is a C1 to C7 straight chain or branched                    chain alkyl optionally substituted with an hydroxyl                    or a C1 to C4 alkoxy group and R₂ is a C1 to C10                    straight chain or branched chain alkyl, alkenyl,                    alkynyl, aryl, alkylaryl, alkylheteroaryl,                    heteroaryl, (CH₂CH₂O)_(n), or (CH₂CH(CH₃)—O)_(n)H                    and n is 1 to 10;

            -   (iv) one or more acylated monosaccharides, acylated                disaccharides, or acylated polysaccharides; and

            -   (v) any combination of (i) through (iv);

        -   (2) a source of peroxygen; and

        -   (3) an enzyme catalyst comprising a polypeptide, said            polypeptide comprising perhydrolytic activity having the            amino acid sequence of SEQ ID NO: 34; and

    -   (b) at least one peroxycarboxylic acid formed upon combining the        set of reaction components of (a).

The present process produces the desired peroxycarboxylic acid uponcombining the reaction components. The reaction components may remainseparated until use.

In a further aspect, a peroxycarboxylic acid generation and deliverysystem is provided comprising:

-   -   (a) a first compartment comprising        -   (1) an enzyme catalyst comprising a polypeptide having            perhydrolytic activity, said polypeptide comprising the            amino acid sequence of SEQ ID NO: 34;        -   (2) at least one substrate selected from the group            consisting of:            -   (i) one or more esters having the structure

[X]_(m)R₅

-   -   -   -   -   wherein                -   X=an ester group of the formula R₆—C(O)O;                -   R₆═C1 to C7 linear, branched or cyclic hydrocarbyl                    moiety, optionally substituted with hydroxyl groups                    or C1 to C4 alkoxy groups, wherein R₆ optionally                    comprises one or more ether linkages for R₆═C2 to                    C7;                -   R₅=a C1 to C6 linear, branched, or cyclic                    hydrocarbyl moiety or a five-membered cyclic                    heteroaromatic moiety or six-membered cyclic                    aromatic or heteroaromatic moiety optionally                    substituted with hydroxyl groups; wherein each                    carbon atom in R₅ individually comprises no more                    than one hydroxyl group or no more than one ester                    group or carboxylic acid group; wherein R₅                    optionally comprises one or more ether linkages;                -   m=is an integer ranging from 1 to the number of                    carbon atoms in R₅; and                -   wherein said esters have solubility in water of at                    least 5 ppm at 25° C.;

            -   (ii) one or more glycerides having the structure

-   -   -   -   -   wherein R₁═C1 to C21 straight chain or branched                    chain alkyl optionally substituted with an hydroxyl                    or a C1 to C4 alkoxy group and R₃ and R₄ are                    individually H or R₁C(O);

            -   (iii) one or more esters of the formula:

-   -   -   -   -   wherein R₁ is a C1 to C7 straight chain or branched                    chain alkyl optionally substituted with an hydroxyl                    or a C1 to C4 alkoxy group and R₂ is a C1 to C10                    straight chain or branched chain alkyl, alkenyl,                    alkynyl, aryl, alkylaryl, alkylheteroaryl,                    heteroaryl, (CH₂CH₂O)_(n), or (CH₂CH(CH₃)—O)_(n)H                    and n is 1 to 10;

            -   (iv) one or more acylated monosaccharides, acylated                disaccharides, or acylated polysaccharides; and

            -   (v) any combination of (i) through (iv); and

        -   (3) an optional buffer; and

    -   (b) a second compartment comprising        -   (1) source of peroxygen;        -   (2) a peroxide stabilizer; and        -   (3) an optional buffer.

In a further embodiment, a laundry care composition is providedcomprising a polypeptide comprising the amino acid sequence of SEQ IDNO: 34.

In a further embodiment, a personal care composition is providedcomprising a polypeptide comprising the amino acid sequence of SEQ IDNO: 34.

BRIEF DESCRIPTION OF THE BIOLOGICAL SEQUENCES

The following sequences comply with 37 C.F.R. §§1.821-1.825(“Requirements for Patent Applications Containing Nucleotide Sequencesand/or Amino Acid Sequence Disclosures—the Sequence Rules”) and areconsistent with World Intellectual Property Organization (WIPO) StandardST.25 (2009) and the sequence listing requirements of the EuropeanPatent Convention (EPC) and the Patent Cooperation Treaty (PCT) Rules5.2 and 49.5(a-bis), and Section 208 and Annex C of the AdministrativeInstructions. The symbols and format used for nucleotide and amino acidsequence data comply with the rules set forth in 37 C.F.R. §1.822.

SEQ ID NO: 1 is the nucleic acid sequence of the codon-optimized codingregion encoding the wild-type Thermotoga maritima acetyl xylan esterasehaving perhydrolytic activity.

SEQ ID NO: 2 is the amino acid sequence of the wild-type Thermotogamaritima acetyl xylan esterase having perhydrolytic activity.

SEQ ID NOs: 3 and 4 are the nucleic acid sequences of primers used toprepare the C277T variant acetyl xylan esterase.

SEQ ID NO: 5 is the amino acid sequence of the C277T variant acetylxylan esterase having perhydrolytic activity (Published U.S. PatentApplication No. 2010-0087529 to DiCosimo et al.).

SEQ ID NO: 6 is the nucleic acid sequence of the plasmid pSW202/C277T.

SEQ ID NOs: 7 and 8 are the nucleic acid sequences of primers used forerror-prone PCR.

SEQ ID NO: 9 is the nucleic acid sequence encoding the variant acetylxylan esterase 006D11 having the following substitutions relative to thewild-type Thermotoga maritima acetyl xylan esterase amino acid sequence:W28R/P84S/S201G/E243D/C277T.

SEQ ID NO: 10 is the amino acid sequence of the 006D11 variant acetylxylan esterase.

SEQ ID NO: 11 is the nucleic acid sequence encoding the variant acetylxylan esterase 006F12 having the following substitutions relative to thewild-type Thermotoga maritima acetyl xylan esterase amino acid sequence:R261S/I264F/C277T.

SEQ ID NO: 12 is the amino acid sequence of the 006F12 variant acetylxylan esterase.

SEQ ID NO: 13 is the nucleic acid sequence encoding the variant acetylxylan esterase 006D10 having the following substitutions relative to thewild-type Thermotoga maritima acetyl xylan esterase amino acid sequence:W28C/L32P/D151E/C277T.

SEQ ID NO: 14 is the amino acid sequence of the 006D10 variant acetylxylan esterase.

SEQ ID NO: 15 is the nucleic acid sequence encoding the variant acetylxylan esterase 006B12 having the following substitutions relative to thewild-type Thermotoga maritima acetyl xylan esterase amino acid sequence:W28C/F104S/C277T.

SEQ ID NO: 16 is the amino acid sequence of the 006B12 variant acetylxylan esterase.

SEQ ID NO: 17 is the nucleic acid sequence encoding the variant acetylxylan esterase 006E11 having the following substitutions relative to thewild-type Thermotoga maritima acetyl xylan esterase amino acid sequence:V183D/C277T.

SEQ ID NO: 18 is the amino acid sequence of the 006E11 variant acetylxylan esterase.

SEQ ID NO: 19 is the nucleic acid sequence encoding the variant acetylxylan esterase 006H5 having the following substitutions relative to thewild-type Thermotoga maritima acetyl xylan esterase amino acid sequence:C277T/L318P.

SEQ ID NO: 20 is the amino acid sequence of the 006H5 variant acetylxylan esterase.

SEQ ID NO: 21 is the nucleic acid sequence encoding the variant acetylxylan esterase 006B5 having the following substitutions relative to thewild-type Thermotoga maritima acetyl xylan esterase amino acid sequence:P265L/C277T/1297N.

SEQ ID NO: 22 is the amino acid sequence of the 006B5 variant acetylxylan esterase.

SEQ ID NO: 23 is the nucleic acid sequence encoding the variant acetylxylan esterase 006F3 having the following substitutions relative to thewild-type Thermotoga maritima acetyl xylan esterase amino acid sequence:P16L/F38S/T53N/R159CN183D/C277T.

SEQ ID NO: 24 is the amino acid sequence of the 006F3 variant acetylxylan esterase.

SEQ ID NO: 25 is the nucleic acid sequence encoding the variant acetylxylan esterase 006G10 having the following substitutions relative to thewild-type Thermotoga maritima acetyl xylan esterase amino acid sequence:R15H/Q176H/L208Q/C277T.

SEQ ID NO: 26 is the amino acid sequence of the 006G10 variant acetylxylan esterase.

SEQ ID NO: 27 is the nucleic acid sequence encoding the variant acetylxylan esterase 006F1 having the following substitutions relative to thewild-type Thermotoga maritima acetyl xylan esterase amino acid sequence:L32P/C277T.

SEQ ID NO: 28 is the amino acid sequence of the 006F1 variant acetylxylan esterase.

SEQ ID NO: 29 is the nucleic acid sequence encoding the variant acetylxylan esterase 006A10 having the following substitutions relative to thewild-type Thermotoga maritima acetyl xylan esterase amino acid sequence:F268S/C277T.

SEQ ID NO: 30 is the amino acid sequence of the 006A10 variant acetylxylan esterase.

SEQ ID NO: 31 is the nucleic acid sequence encoding the variant acetylxylan esterase 006G6 having the following substitutions relative to thewild-type Thermotoga maritima acetyl xylan esterase amino acid sequence:E29G/S62P/F174L/C277T.

SEQ ID NO: 32 is the amino acid sequence of the 006G6 variant acetylxylan esterase.

SEQ ID NO: 33 is the nucleic acid sequence encoding the variant acetylxylan esterase 006E10 having the following substitutions relative to thewild-type Thermotoga maritima acetyl xylan esterase amino acid sequence:R218C/C277T/F317L.

SEQ ID NO: 34 is the amino acid sequence of the 006E10 variant acetylxylan esterase.

SEQ ID NO: 35 is the nucleic acid sequence encoding the variant acetylxylan esterase 006D9 having the following substitutions relative to thewild-type Thermotoga maritima acetyl xylan esterase amino acid sequence:C277T/R296P.

SEQ ID NO: 36 is the amino acid sequence of the 006D9 variant acetylxylan esterase.

SEQ ID NO: 37 is the nucleic acid sequence encoding the variant acetylxylan esterase 006F7 having the following substitutions relative to thewild-type Thermotoga maritima acetyl xylan esterase amino acid sequence:E9K/G255C/C277T.

SEQ ID NO: 38 is the amino acid sequence of the 006F7 variant acetylxylan esterase.

SEQ ID NO: 39 is the nucleic acid sequence encoding the variant acetylxylan esterase 006E12 having the following substitutions relative to thewild-type Thermotoga maritima acetyl xylan esterase amino acid sequence:H227UT233A/C277T/A290V.

SEQ ID NO: 40 is the amino acid sequence of the 006E12 variant acetylxylan esterase.

SEQ ID NO: 41 is the nucleic acid sequence encoding the variant acetylxylan esterase 006F8 having the following substitutions relative to thewild-type Thermotoga maritima acetyl xylan esterase amino acid sequence:Q67R/K82E/C277T.

SEQ ID NO: 42 is the amino acid sequence of the 006F8 variant acetylxylan esterase.

SEQ ID NO: 43 is the nucleic acid sequence encoding the variant acetylxylan esterase 006G8 having the following substitutions relative to thewild-type Thermotoga maritima acetyl xylan esterase amino acid sequence:F247L/C277T/V282D.

SEQ ID NO: 44 is the amino acid sequence of the 006G8 variant acetylxylan esterase.

SEQ ID NO: 45 is the nucleic acid sequence encoding the variant acetylxylan esterase 006G11 having the following substitutions relative to thewild-type Thermotoga maritima acetyl xylan esterase amino acid sequence:D254G/C277T.

SEQ ID NO: 46 is the amino acid sequence of the 006G11 variant acetylxylan esterase.

SEQ ID NO: 47 is the nucleic acid sequence encoding the variant acetylxylan esterase 006E9 having the following substitutions relative to thewild-type Thermotoga maritima acetyl xylan esterase amino acid sequence:E17V/R261H/C277T.

SEQ ID NO: 48 is the amino acid sequence of the 006E9 variant acetylxylan esterase.

SEQ ID NO: 49 is the nucleic acid sequence encoding the variant acetylxylan esterase 006F10 having the following substitutions relative to thewild-type Thermotoga maritima acetyl xylan esterase amino acid sequence:F24L/T31S/E48V/C277T/K316E.

SEQ ID NO: 50 is the amino acid sequence of the 006F10 variant acetylxylan esterase.

SEQ ID NO: 51 is the nucleic acid sequence encoding the variant acetylxylan esterase 006F5 having the following substitutions relative to thewild-type Thermotoga maritima acetyl xylan esterase amino acid sequence:V43G/G63C/H100L/C277T/A284V.

SEQ ID NO: 52 is the amino acid sequence of the 006F5 variant acetylxylan esterase.

SEQ ID NO: 53 is the nucleic acid sequence encoding the variant acetylxylan esterase 006E7 having the following substitutions relative to thewild-type Thermotoga maritima acetyl xylan esterase amino acid sequence:F258Y/C277T1E294G/L318P.

SEQ ID NO: 54 is the amino acid sequence of the 006E7 variant acetylxylan esterase.

SEQ ID NO: 55 is the nucleic acid sequence encoding the variant acetylxylan esterase 006G12 having the following substitutions relative to thewild-type Thermotoga maritima acetyl xylan esterase amino acid sequence:F161L/C277T.

SEQ ID NO: 56 is the amino acid sequence of the 006G12 variant acetylxylan esterase.

SEQ ID NO: 57 is the nucleic acid sequence encoding the variant acetylxylan esterase 006H01 having the following substitutions relative to thewild-type Thermotoga maritima acetyl xylan esterase amino acid sequence:F253S/C277T.

SEQ ID NO: 58 is the amino acid sequence of the 006H01 variant acetylxylan esterase.

SEQ ID NO: 59 is the nucleic acid sequence encoding the variant acetylxylan esterase 006G2 having the following substitutions relative to thewild-type Thermotoga maritima acetyl xylan esterase amino acid sequence:F24I/C277T.

SEQ ID NO: 60 is the amino acid sequence of the 006G2 variant acetylxylan esterase.

SEQ ID NO: 61 is the nucleic acid sequence encoding the variant acetylxylan esterase 006F6 having the following substitutions relative to thewild-type Thermotoga maritima acetyl xylan esterase amino acid sequence:A206T/C277T.

SEQ ID NO: 62 is the amino acid sequence of the 006F6 variant acetylxylan esterase.

SEQ ID NO: 63 is the nucleic acid sequence encoding the variant acetylxylan esterase 006C10 having the following substitutions relative to thewild-type Thermotoga maritima acetyl xylan esterase amino acid sequence:E55K/C277T.

SEQ ID NO: 64 is the amino acid sequence of the 006C10 variant acetylxylan esterase.

SEQ ID NO: 65 is the nucleic acid sequence encoding the variant acetylxylan esterase 006F9 having the following substitutions relative to thewild-type Thermotoga maritima acetyl xylan esterase amino acid sequence:Q67R/C277T.

SEQ ID NO: 66 is the amino acid sequence of the 006F9 variant acetylxylan esterase.

SEQ ID NOs: 67 and 68 are the nucleic acid sequences of primers used toprepare the C277S variant acetyl xylan esterase.

SEQ ID NO: 69 is the amino acid sequence of the C277S variant acetylxylan esterase having perhydrolytic activity (U.S. patent applicationSer. No. 12/572,094 to DiCosimo et al.).

SEQ ID NO: 70 is the nucleic acid sequence of the plasmid pSW202/C277S.

SEQ ID NOs: 71-82 are primers.

SEQ ID NO: 83 is the nucleic acid sequence encoding the variant acetylxylan esterase 873C8 having the following substitutions relative to thewild-type Thermotoga maritima acetyl xylan esterase amino acid sequence:S35R/C277S.

SEQ ID NO: 84 is the amino acid sequence of the 873C8 variant acetylxylan esterase.

SEQ ID NO: 85 is the nucleic acid sequence encoding the variant acetylxylan esterase 873D7 having the following substitutions relative to thewild-type Thermotoga maritima acetyl xylan esterase amino acid sequence:S35K/C277S.

SEQ ID NO: 86 is the amino acid sequence of the 873D7 variant acetylxylan esterase.

SEQ ID NO: 87 is the nucleic acid sequence encoding the variant acetylxylan esterase 873D3 having the following substitutions relative to thewild-type Thermotoga maritima acetyl xylan esterase amino acid sequence:S35E/C277S.

SEQ ID NO: 88 is the amino acid sequence of the 873D3 variant acetylxylan esterase.

SEQ ID NO: 89 is the nucleic acid sequence encoding the variant acetylxylan esterase 873C9 having the following substitutions relative to thewild-type Thermotoga maritima acetyl xylan esterase amino acid sequence:S35N/C277S.

SEQ ID NO: 90 is the amino acid sequence of the 873C9 variant acetylxylan esterase.

SEQ ID NO: 91 is the nucleic acid sequence encoding the variant acetylxylan esterase 873H5 having the following substitutions relative to thewild-type Thermotoga maritima acetyl xylan esterase amino acid sequence:K77A/C277S.

SEQ ID NO: 92 is the amino acid sequence of the 873H5 variant acetylxylan esterase.

SEQ ID NO: 93 is the nucleic acid sequence encoding the variant acetylxylan esterase 873G12 having the following substitutions relative to thewild-type Thermotoga maritima acetyl xylan esterase amino acid sequence:K77V/C277S.

SEQ ID NO: 94 is the amino acid sequence of the 873G12 variant acetylxylan esterase.

SEQ ID NO: 95 is the nucleic acid sequence encoding the variant acetylxylan esterase 873H1 having the following substitutions relative to thewild-type Thermotoga maritima acetyl xylan esterase amino acid sequence:K77D/C277S.

SEQ ID NO: 96 is the amino acid sequence of the 873H1 variant acetylxylan esterase.

SEQ ID NO: 97 is the nucleic acid sequence encoding the variant acetylxylan esterase 873A4 having the following substitutions relative to thewild-type Thermotoga maritima acetyl xylan esterase amino acid sequence:L125M/C277S.

SEQ ID NO: 98 is the amino acid sequence of the 873A4 variant acetylxylan esterase.

SEQ ID NO: 99 is the nucleic acid sequence encoding the variant acetylxylan esterase 873A9 having the following substitutions relative to thewild-type Thermotoga maritima acetyl xylan esterase amino acid sequence:L125R/C277S.

SEQ ID NO: 100 is the amino acid sequence of the 873A9 variant acetylxylan esterase.

SEQ ID NO: 101 is the nucleic acid sequence encoding the variant acetylxylan esterase 873B4 having the following substitutions relative to thewild-type Thermotoga maritima acetyl xylan esterase amino acid sequence:A266P/C277S.

SEQ ID NO: 102 is the amino acid sequence of the 873B4 variant acetylxylan esterase.

SEQ ID NO: 103 is the nucleic acid sequence encoding the variant acetylxylan esterase 873B2 having the following substitutions relative to thewild-type Thermotoga maritima acetyl xylan esterase amino acid sequence:A266D/C277S.

SEQ ID NO: 104 is the amino acid sequence of the 873B2 variant acetylxylan esterase.

SEQ ID NO: 105 is the nucleic acid sequence encoding the variant acetylxylan esterase 873G7 having the following substitutions relative to thewild-type Thermotoga maritima acetyl xylan esterase amino acid sequence:A266I/C277S.

SEQ ID NO: 106 is the amino acid sequence of the 873G7 variant acetylxylan esterase.

SEQ ID NO: 107 is the nucleic acid sequence encoding the variant acetylxylan esterase 873C3 having the following substitutions relative to thewild-type Thermotoga maritima acetyl xylan esterase amino acid sequence:A266E/C277S.

SEQ ID NO: 108 is the amino acid sequence of the 873C3 variant acetylxylan esterase.

SEQ ID NO: 109 is the nucleic acid sequence encoding the variant acetylxylan esterase 873B3 having the following substitutions relative to thewild-type Thermotoga maritima acetyl xylan esterase amino acid sequence:A266N/C277S.

SEQ ID NO: 110 is the amino acid sequence of the 873B3 variant acetylxylan esterase.

SEQ ID NO: 111 is the nucleic acid sequence encoding the variant acetylxylan esterase 873B12 having the following substitutions relative to thewild-type Thermotoga maritima acetyl xylan esterase amino acid sequence:A266R/C277S.

SEQ ID NO: 112 is the amino acid sequence of the 873612 variant acetylxylan esterase.

SEQ ID NO: 113 is the nucleic acid sequence encoding the variant acetylxylan esterase 873G3 having the following substitutions relative to thewild-type Thermotoga maritima acetyl xylan esterase amino acid sequence:A266Q/C277S.

SEQ ID NO: 114 is the amino acid sequence of the 873G3 variant acetylxylan esterase.

SEQ ID NO: 115 is the nucleic acid sequence encoding the variant acetylxylan esterase 873E2 having the following substitutions relative to thewild-type Thermotoga maritima acetyl xylan esterase amino acid sequence:A266V/C277S.

SEQ ID NO: 116 is the amino acid sequence of the 873E2 variant acetylxylan esterase.

DETAILED DESCRIPTION

A nucleic acid molecule encoding the Thermotoga maritima C277T variantacetyl xylan esterase (SEQ ID NO: 5) was mutated to create a library ofvariant perhydrolases. Several perhydrolase variants were identifiedexhibiting an increase in specific activity when compared to thespecific activity of the Thermotoga maritima C277T perhydrolase havingamino acid sequence SEQ ID NO: 5.

A library of variant perhydrolases was also created using sitesaturation mutagenesis of the Thermotoga maritima variant acetyl xylanesterase C277S (SEQ ID NO: 69) at residues S25, K77, L125M, A266, N275,and N₃O₂. A perhydrolase variant was identified exhibiting an increasein specific activity when compared to the specific activity of theThermotoga maritima C277S perhydrolase having amino acid sequence SEQ IDNO: 69.

Compositions and methods are provided comprising the variantperhydrolase enzyme having amino acid sequence SEQ ID NO: 34.

In this disclosure, a number of terms and abbreviations are used. Thefollowing definitions apply unless specifically stated otherwise.

As used herein, the articles “a”, “an”, and “the” preceding an elementor component of the invention are intended to be nonrestrictiveregarding the number of instances (i.e., occurrences) of the element orcomponent Therefore “a”, “an” and “the” should be read to include one orat least one, and the singular word form of the element or componentalso includes the plural unless the number is obviously meant to besingular.

The term “comprising” means the presence of the stated features,integers, steps, or components as referred to in the claims, but doesnot preclude the presence or addition of one or more other features,integers, steps, components or groups thereof. The term “comprising” isintended to include embodiments encompassed by the terms “consistingessentially of” and “consisting of”. Similarly, the term “consistingessentially of” is intended to include embodiments encompassed by theterm “consisting of”.

As used herein, the term “about” modifying the quantity of an ingredientor reactant employed refers to variation in the numerical quantity thatcan occur, for example, through typical measuring and liquid handlingprocedures used for making concentrates or use solutions in the realworld; through inadvertent error in these procedures; throughdifferences in the manufacture, source, or purity of the ingredientsemployed to make the compositions or carry out the methods; and thelike. The term “about” also encompasses amounts that differ due todifferent equilibrium conditions for a composition resulting from aparticular initial mixture. Whether or not modified by the term “about”,the claims include equivalents to the quantities.

Where present, all ranges are inclusive and combinable. For example,when a range of “1 to 5” is recited, the recited range should beconstrued as including ranges “1 to 4”, “1 to 3”, “1-2”, “1-2 & 4-5”,“1-3 & 5”, and the like.

As used herein, the term “multi-component system” will refer to a systemof enzymatically generating peroxycarboxylic acid wherein the componentsremain separated until use. As such, the multi-component system willinclude at least one first component that remains separated from atleast one second component. The first and second components areseparated in different compartments until use (i.e., using first andsecond compartments). The design of the multi-component systems willoften depend on the physical form of the components to be combined andare described in more detail below.

As used herein, the term “peroxycarboxylic acid” is synonymous withperacid, peroxyacid, peroxy acid, percarboxylic acid and peroxoic acid.

As used herein, the term “peracetic acid” is abbreviated as “PAA” and issynonymous with peroxyacetic acid, ethaneperoxoic acid and all othersynonyms of CAS Registry Number 79-21-0.

As used herein, the term “monoacetin” is synonymous with glycerolmonoacetate, glycerin monoacetate, and glyceryl monoacetate.

As used herein, the term “diacetin” is synonymous with glyceroldiacetate; glycerin diacetate, glyceryl diacetate, and all othersynonyms of CAS Registry Number 25395-31-7.

As used herein, the term “triacetin” is synonymous with glycerintriacetate; glycerol triacetate; glyceryl triacetate,1,2,3-triacetoxypropane, 1,2,3-propanetriol triacetate and all othersynonyms of CAS Registry Number 102-76-1.

As used herein, the term “monobutyrin” is synonymous with glycerolmonobutyrate, glycerin monobutyrate, and glyceryl monobutyrate.

As used herein, the term “dibutyrin” is synonymous with glyceroldibutyrate and glyceryl dibutyrate.

As used herein, the term “tributyrin” is synonymous with glyceroltributyrate, 1,2,3-tributyrylglycerol, and all other synonyms of CASRegistry Number 60-01-5.

As used herein, the term “monopropionin” is synonymous with glycerolmonopropionate, glycerin monopropionate, and glyceryl monopropionate.

As used herein, the term “dipropionin” is synonymous with glyceroldipropionate and glyceryl dipropionate.

As used herein, the term “tripropionin” is synonymous with glyceryltripropionate, glycerol tripropionate, 1,2,3-tripropionylglycerol, andall other synonyms of CAS Registry Number 139-45-7.

As used herein, the term “ethyl acetate” is synonymous with aceticether, acetoxyethane, ethyl ethanoate, acetic acid ethyl ester, ethanoicacid ethyl ester, ethyl acetic ester and all other synonyms of CASRegistry Number 141-78-6.

As used herein, the term “ethyl lactate” is synonymous with lactic acidethyl ester and all other synonyms of CAS Registry Number 97-64-3.

As used herein, the terms “acylated sugar” and “acylated saccharide”refer to mono-, di- and polysaccharides comprising at least one acylgroup, where the acyl group is selected from the group consisting ofstraight chain aliphatic carboxylates having a chain length from C2 toC8. Examples include, but are not limited to, glucose pentaacetate,xylose tetraacetate, acetylated xylan, acetylated xylan fragments,β-D-ribofuranose-1,2,3,5-tetraacetate, tri-O-acetyl-D-galactal, andtri-O-acetyl-glucal.

As used herein, the terms “hydrocarbyl”, “hydrocarbyl group”, and“hydrocarbyl moiety” mean a straight chain, branched or cyclicarrangement of carbon atoms connected by single, double, or triplecarbon to carbon bonds and/or by ether linkages, and substitutedaccordingly with hydrogen atoms. Such hydrocarbyl groups may bealiphatic and/or aromatic. Examples of hydrocarbyl groups includemethyl, ethyl, propyl, isopropyl, butyl, isobutyl, t-butyl, cyclopropyl,cyclobutyl, pentyl, cyclopentyl, methylcyclopentyl, hexyl, cyclohexyl,benzyl, and phenyl. In one embodiment, the hydrocarbyl moiety is astraight chain, branched or cyclic arrangement of carbon atoms connectedby single carbon to carbon bonds and/or by ether linkages, andsubstituted accordingly with hydrogen atoms.

As used herein, the term “aromatic” refers to an organic compound ormoiety characterized by increased chemical stability resulting from thedelocalization of electrons in a ring system containing usually multipleconjugated double bonds. Planar monocyclic conjugated rings havingdelocalized electrons should be aromatic if the have (4n+2) π electrons.Examples of aromatic compounds may include derivatives of benzene (suchas 2-, 3- or 4-acetoxybenzoic acid). In one embodiment, the estersubstrate may be 4-acetoxybenzoic acid.

As used herein, the term “heterocyclic” refers to an organic compound ormoiety with a ring structure having one or more atoms other than carbonin at least one of its rings.

As used herein, the term “heteroaromatic” refers to an organic compoundor moiety with a ring structure that is both heterocyclic and aromatic,wherein the ring comprises at least one of the heteroatoms oxygen,nitrogen, or sulfur. Examples of heteroaromatic moieties may includepyridine, pyrrole, furan, and thiophene moieties.

As used herein, the terms “monoesters” and “diesters” of 1,2-ethanediol,1,2-propanediol, 1,3-propanediol, 1,2-butanediol, 1,3-butanediol,2,3-butanediol, 1,4-butanediol, 1,2-pentanediol, 2,5-pentanediol,1,6-pentanediol, 1,2-hexanediol, 2,5-hexanediol, 1,6-hexanediol, referto said compounds comprising at least one ester group of the formulaRC(O)O, wherein R is a C1 to C7 linear hydrocarbyl moiety.

As used herein, the terms “suitable enzymatic reaction formulation”,“components suitable for generation of a peroxycarboxylic acid”,“suitable reaction components”, “reaction components”, “reactionformulation”, and “suitable aqueous reaction formulation” refer to thematerials and water in which the reactants and the enzyme catalystcomprising the present variant polypeptide having perhydrolytic activitycome into contact to form the desired peroxycarboxylic acid. Thecomponents of the reaction formulation are provided herein and thoseskilled in the art appreciate the range of component variations suitablefor this process. In one embodiment, the enzymatic reaction formulationproduces peroxycarboxylic acid in situ upon combining the reactioncomponents. As such, the reaction components may be provided as amulti-component system wherein one or more of the reaction componentsremains separated until use. The design of systems and means forseparating and combining multiple active components are known in the artand generally will depend upon the physical form of the individualreaction components. For example, multiple active fluids (liquid-liquid)systems typically use multi-chamber dispenser bottles or two-phasesystems (U.S. Patent Application Publication No. 2005-0139608; U.S. Pat.No. 5,398,846; U.S. Pat. No. 5,624,634; U.S. Pat. No. 6,391,840; E.P.Patent 0807156B1; U.S. Patent Application Publication No. 2005-0008526;and PCT Publication No. WO 00/61713A1) such as found in some bleachingapplications wherein the desired bleaching agent is produced upon mixingthe reactive fluids. Multi-component formulations and multi-componentgeneration systems to enzymatically produce peroxycarboxylic acids fromcarboxylic acid esters are described by DiCosimo et al., in PublishedU.S. Patent Application Nos. 2010-0086510 and 2010-0086621,respectively. Other forms of multi-component systems used to generateperoxycarboxylic acid may include, but are not limited to, thosedesigned for one or more solid components or combinations ofsolid-liquid components, such as powders used in many commerciallyavailable bleaching compositions (e.g., U.S. Pat. No. 5,116,575),multi-layered tablets (e.g., U.S. Pat. No. 6,210,639), water dissolvablepackets having multiple compartments (e.g., U.S. Pat. No. 6,995,125) andsolid agglomerates that react upon the addition of water (e.g., U.S.Pat. No. 6,319,888).

As used herein, the term “substrate” or “carboxylic acid estersubstrate” will refer to the reaction components enzymaticallyperhydrolyzed using the present enzyme catalyst in the presence of asuitable source of peroxygen, such as hydrogen peroxide. In oneembodiment, the substrate comprises at least one ester group capable ofbeing enzymatically perhydrolyzed using the enzyme catalyst, whereby aperoxycarboxylic acid is produced.

As used herein, the term “perhydrolysis” is defined as the reaction of aselected substrate with a source of hydrogen peroxide to form aperoxycarboxylic acid. Typically, inorganic peroxide is reacted with theselected substrate in the presence of a catalyst to produce theperoxycarboxylic acid. As used herein, the term “chemical perhydrolysis”includes perhydrolysis reactions in which a substrate (such as aperoxycarboxylic acid precursor) is combined with a source of hydrogenperoxide wherein peroxycarboxylic acid is formed in the absence of anenzyme catalyst. As used herein, the term “enzymatic perhydrolysis”refers a reaction of a selected substrate with a source of hydrogenperoxide to form a peroxycarboxylic acid, wherein the reaction iscatalyzed by an enzyme catalyst having perhydrolysis activity.

As used herein, the term “perhydrolase activity” refers to the enzymecatalyst activity per unit mass (for example, milligram) of protein, drycell weight, or immobilized catalyst weight.

As used herein, “one unit of enzyme activity” or “one unit of activity”or “U” is defined as the amount of perhydrolase activity required forthe production of 1 μmol of peroxycarboxylic acid product (such asperacetic acid) per minute at a specified temperature. “One unit ofenzyme activity” may also be used herein to refer to the amount ofperoxycarboxylic acid hydrolysis activity required for the hydrolysis of1 μmol of peroxycarboxylic acid (e.g., peracetic acid) per minute at aspecified temperature.

The present variant CE-7 carbohydrate esterase is characterized by anincrease in specific activity when compared to the perhydrolase fromwhich it was derived (Thermotoga maritima C277T; Published U.S. PatentApplication No. 2010-0087529) under the same reaction conditions. Asused herein, the “fold increase” in specific activity is measuredrelative to the specific activity of the parent perhydrolase from whichthe variant was derived (either the Thermotoga maritima C277Tperhydrolase (SEQ ID NO: 5) or Thermotoga maritima C277S perhydrolase(SEQ ID NO: 69)) under the same reaction conditions. In one embodiment,the fold increase in specific activity of the variant polypeptide (i.e.,variant perhydrolase) relative to the parent perhydrolase (Thermotogamaritima C277T or C277S) is at least 1.01, 1.05, 1.1, 1.2, 1.3, 1.4,1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 3.0, 4.0, 5.0, 6.0, 7.0, 8.0, 9.0, or10-fold when compared under identical reaction/assay conditions.

As used herein, “identical assay conditions” or “same assay conditions”refer to the conditions used to measure the peracid formation (i.e.,perhydrolysis of a carboxylic acid ester substrate) specific activity ofthe variant polypeptide relative to the respective specific activity ofthe polypeptide from which it is was derived. The assay conditions usedto measure the respective specific activities should be as close toidentical as possible such that only the structure of the polypeptidehaving perhydrolytic activity varies.

As used herein, the terms “enzyme catalyst” and “perhydrolase catalyst”refer to a catalyst comprising an enzyme (i.e., a polypeptide) havingperhydrolysis activity and may be in the form of a whole microbial cell,permeabilized microbial cell(s), one or more cell components of amicrobial cell extract, partially purified enzyme, or purified enzyme.The enzyme catalyst may also be chemically modified (for example, bypegylation or by reaction with cross-linking reagents). The perhydrolasecatalyst may also be immobilized on a soluble or insoluble support usingmethods well-known to those skilled in the art; see for example,Immobilization of Enzymes and Cells; Gordon F. Bickerstaff, Editor;Humana Press, Totowa, N.J., USA; 1997.

The present enzyme catalyst comprises a variant polypeptide havingperhydrolytic activity and is structurally classified as a member of thecarbohydrate family esterase family 7 (CE-7 family) of enzymes (seeCoutinho, P. M., Henrissat, B. “Carbohydrate-active enzymes: anintegrated database approach” in Recent Advances in CarbohydrateBioengineering, H. J. Gilbert, G. Davies, B. Henrissat and B. Svenssoneds., (1999) The Royal Society of Chemistry, Cambridge, pp. 3-12.). TheCE-7 family of enzymes has been demonstrated to be particularlyeffective for producing peroxycarboxylic acids from a variety ofcarboxylic acid ester substrates when combined with a source ofperoxygen (See PCT publication No. WO2007/070609 and U.S. PatentApplication Publication No. 2008-0176299 and U.S. Pat. Nos. 7,951,566and 7,723,083 to DiCosimo et al.; each herein incorporated by referencein their entireties). The CE-7 enzyme family includes cephalosporin Cdeacetylases (CAHs; E.C. 3.1.1.41) and acetyl xylan esterases (AXEs;E.C. 3.1.1.72). Members of the CE-7 enzyme family share a conservedsignature motif (Vincent et al., J. Mol. Biol., 330:593-606 (2003)).

As used herein, the terms “signature motif” and “CE-7 signature motif”,refer to conserved structures shared among a family of enzymes having aperhydrolytic activity.

As used herein, “structurally classified as a CE-7 enzyme”,“structurally classified as a carbohydrate esterase family 7 enzyme”,“structurally classified as a CE-7 carbohydrate esterase”, and “CE-7perhydrolase” will be used to refer to enzymes having perhydrolysisactivity that are structurally classified as a CE-7 carbohydrateesterase based on the presence of the CE-7 signature motif (Vincent etal., supra). The “signature motif” for CE-7 esterases comprises threeconserved motifs (residue position numbering relative to referencesequence SEQ ID NO: 2; the wild-type Thermotoga maritima acetyl xylanesterase):

a) Arg118-Gly119-Gln120;

b) Gly186-Xaa187-Ser188-Gln189-Gly190; and

c) His303-Glu304.

Typically, the Xaa at amino acid residue position 187 is glycine,alanine, proline, tryptophan, or threonine. Two of the three amino acidresidues belonging to the catalytic triad are in bold. In oneembodiment, the Xaa at amino acid residue position 187 is selected fromthe group consisting of glycine, alanine, proline, tryptophan, andthreonine.

Further analysis of the conserved motifs within the CE-7 carbohydrateesterase family indicates the presence of an additional conserved motif(LXD at amino acid positions 272-274 of SEQ ID NO: 2) that may be usedto further define a member of the CE-7 carbohydrate esterase family. Ina further embodiment, the signature motif defined above includes afourth conserved motif defined as:

Leu272-Xaa273-Asp274.

The Xaa at amino acid residue position 273 is typically isoleucine,valine, or methionine. The fourth motif includes the aspartic acidresidue (bold) belonging to the catalytic triad (Ser188-Asp274-His303).

As used herein, the terms “cephalosporin C deacetylase” and“cephalosporin C acetyl hydrolase” refer to an enzyme (E.C. 3.1.1.41)that catalyzes the deacetylation of cephalosporins such as cephalosporinC and 7-aminocephalosporanic acid (Mitsushima et al., Appl. Environ.Microbiol., 61(6): 2224-2229 (1995); U.S. Pat. No. 5,528,152; and U.S.Pat. No. 5,338,676). Enzymes classified as cephalosporin C deacetylaseshave been shown to often have significant perhydrolytic activity (U.S.Pat. No. 7,951,566 and U.S. Patent Application Publication No.2008-0176299 to DiCosimo et al.).

As used herein, “acetyl xylan esterase” refers to an enzyme (E.C.3.1.1.72; AXEs) that catalyzes the deacetylation of acetylated xylansand other acetylated saccharides. Enzymes classified as acetyl xylanesterases have been shown to have significant perhydrolytic activity(U.S. Pat. Nos. 7,951,566 and 7,723,083 and U.S. Patent ApplicationPublication No. 2008-0176299, each to DiCosimo et al.).

As used herein, the term “Thermotoga maritima” refers to a bacterialcell reported to have acetyl xylan esterase activity(GENBANK®NP_(—)227893.1). In one aspect, the Thermotoga maritima strainis Thermotoga maritima MSB8. The amino acid sequence of the wild-typeenzyme having perhydrolase activity from Thermotoga maritima is providedas SEQ ID NO: 2.

As used herein, the terms “variant”, “variant polypeptide”, and “variantenzyme catalyst” refer to an enzyme catalyst comprising at least onepolypeptide (i.e., a perhydrolase) having perhydrolytic activity whereinthe polypeptide comprises at least one amino acid change relative to theenzyme/polypeptide from which it was derived (i.e., Thermotoga maritimaC277S or C277T perhydrolase). Several variant polypeptides are providedherein having perhydrolytic activity and are characterized by anincrease in specific activity relative to the Thermotoga maritima C277Tacetyl xylan esterase having amino acid sequence SEQ ID NO: 5. A variantpolypeptide having an increase in perhydrolytic specific activityrelative to the Thermotoga maritima C277S (SEQ ID NO: 69) is alsoprovided (Published U.S. Patent Application No. 2010-0087529 and U.S.patent application Ser. No. 13/070,123).

For a particular variant perhydrolase, amino acid substitutions arespecified with reference to the wild type Thermotoga maritima amino acidsequence (SEQ ID NO: 2). The wild-type amino acid (denoted by thestandard single letter abbreviation) is followed by the amino acidresidue position of SEQ ID NO: 2 followed by the amino acid of thevariant (also denoted by the standard single letter abbreviation). Forexample, “C277S” describes a change in SEQ ID NO: 2 at amino acidresidue position 277 where cysteine was changed to serine. The variantpolypeptide may be comprised of multiple point substitutions. Forexample, R261S/I264F/C277T refers to a variant polypeptide having threepoint substitutions: 1) a change at amino acid residue position 261where an arginine was changed to a serine, 2) a change at residueposition 264 where an isoleucine was changed to a phenylalanine, and 3)a change at position 277 where a cysteine was changed to a threonine.

The term “amino acid” refers to the basic chemical structural unit of aprotein or polypeptide. The following abbreviations are used herein toidentify specific amino adds:

Three-Letter One-Letter Amino Acid Abbreviation Abbreviation Alanine AlaA Arginine Arg R Asparagine Asn N Aspartic acid Asp D Cysteine Cys CGlutamine Gln Q Glutamic acid Glu E Glycine Gly G Histidine His HIsoleucine Ile I Leucine Leu L Lysine Lys K Methionine Met MPhenylalanine Phe F Proline Pro P Serine Ser S Threonine Thr TTryptophan Trp W Tyrosine Tyr Y Valine Val V Any amino acid (or asdefined herein) Xaa X

As used herein, the term “biological contaminants” refers to one or moreunwanted and/or pathogenic biological entities including, but notlimited to microorganisms, spores, viruses, prions, and mixturesthereof. The present enzyme can be used to produce an efficaciousconcentration of at least one peroxycarboxylic acid useful to reduceand/or eliminate the presence of the viable biological contaminants. Ina preferred embodiment, the biological contaminant is a viablepathogenic microorganism.

As used herein, the term “disinfect” refers to the process ofdestruction of or prevention of the growth of biological contaminants.As used herein, the term “disinfectant” refers to an agent thatdisinfects by destroying, neutralizing, or inhibiting the growth ofbiological contaminants. Typically, disinfectants are used to treatinanimate objects or surfaces. As used herein, the term “antiseptic”refers to a chemical agent that inhibits the growth of disease-carryingmicroorganisms. In one aspect of the embodiment, the biologicalcontaminants are pathogenic microorganisms.

As used herein, the term “sanitary” means of or relating to therestoration or preservation of health, typically by removing, preventingor controlling an agent that may be injurious to health. As used herein,the term “sanitize” means to make sanitary. As used herein, the term“sanitizer” refers to a sanitizing agent. As used herein the term“sanitization” refers to the act or process of sanitizing.

As used herein, the term “virucide” refers to an agent that inhibits ordestroys viruses, and is synonymous with “viricide”. An agent thatexhibits the ability to inhibit or destroy viruses is described ashaving “virucidal” activity. Peroxycarboxylic acids can have virucidalactivity. Typical alternative virucides known in the art which may besuitable for use with the present invention include, for example,alcohols, ethers, chloroform, formaldehyde, phenols, beta propiolactone,iodine, chlorine, mercury salts, hydroxylamine, ethylene oxide, ethyleneglycol, quaternary ammonium compounds, enzymes, and detergents.

As used herein, the term “biocide” refers to a chemical agent, typicallybroad spectrum, which inactivates or destroys microorganisms. A chemicalagent that exhibits the ability to inactivate or destroy microorganismsis described as having “biocidal” activity. Peroxycarboxylic acids canhave biocidal activity. Typical alternative biocides known in the art,which may be suitable for use in the present invention include, forexample, chlorine, chlorine dioxide, chloroisocyanurates, hypochlorites,ozone, acrolein, amines, chlorinated phenolics, copper salts,organo-sulphur compounds, and quaternary ammonium salts.

As used herein, the phrase “minimum biocidal concentration” refers tothe minimum concentration of a biocidal agent that, for a specificcontact time, will produce a desired lethal, irreversible reduction inthe viable population of the targeted microorganisms. The effectivenesscan be measured by the log₁₀ reduction in viable microorganisms aftertreatment. In one aspect, the targeted reduction in viablemicroorganisms after treatment is at least a 3−log₁₀ reduction, morepreferably at least a 4−log₁₀ reduction, and most preferably at least a5−log₁₀ reduction. In another aspect, the minimum biocidal concentrationis at least a 6−log₁₀ reduction in viable microbial cells.

As used herein, the terms “peroxygen source” and “source of peroxygen”refer to compounds capable of providing hydrogen peroxide at aconcentration of about 1 mM or more when in an aqueous solutionincluding, but not limited to, hydrogen peroxide, hydrogen peroxideadducts (e.g., urea-hydrogen peroxide adduct (carbamide peroxide)),perborates, and percarbonates, such as sodium percarbonate. As describedherein, the concentration of the hydrogen peroxide provided by theperoxygen compound in the aqueous reaction formulation is initially atleast 1 mM or more upon combining the reaction components. In oneembodiment, the hydrogen peroxide concentration in the aqueous reactionformulation is at least 0.5 mM. In another embodiment, the hydrogenperoxide concentration in the aqueous reaction formulation is at least10 mM. In another embodiment, the hydrogen peroxide concentration in theaqueous reaction formulation is at least 100 mM. In another embodiment,the hydrogen peroxide concentration in the aqueous reaction formulationis at least 200 mM. In another embodiment, the hydrogen peroxideconcentration in the aqueous reaction formulation is 500 mM or more. Inyet another embodiment, the hydrogen peroxide concentration in theaqueous reaction formulation is 1000 mM or more. The molar ratio of thehydrogen peroxide to enzyme substrate, such as triglyceride,(H₂O₂:substrate) in the aqueous reaction formulation may be from about0.002 to 20, preferably about 0.1 to 10, and most preferably about 0.5to 5.

As used herein, the term “benefit agent” refers to a material thatpromotes or enhances a useful advantage, a favorable/desirable effect orbenefit. In one embodiment, a process is provided whereby a benefitagent, such as a composition comprising a peroxycarboxylic acid, isapplied to a textile or article of clothing to achieve a desiredbenefit, such as disinfecting, bleaching, destaining, deodorizing, andany combination thereof. In another embodiment, the present variantpolypeptide having perhydrolytic activity may be used to produce aperacid-based benefit agent for use in personal care products (such ashair care products, skin care products, nail care products or oral careproducts). In one embodiment, a personal care product is providedcomprising the variant perhydrolase having amino acid sequence SEQ IDNO: 34. The personal care products are formulated to provide a safe andefficacious concentration of the desired peracid benefit agent.

Variant Polypeptides Having an Increase in Specific Activity.

The present variant polypeptides were derived from the Thermotogamaritima C277S or Thermotoga maritima C277T acetyl xylan esterase thathas been previously demonstrated to have significant perhydrolyticactivity for producing peroxycarboxylic acids from carboxylic acidesters and a source of peroxygen, such as hydrogen peroxide (U.S. PatentApplication Publication No. 2008-0176299 and 2010-0087529, each toDiCosimo et al.).

Libraries of variant polypeptides were created from the C277T Thermotogamaritima perhydrolase (SEQ ID NO: 5) or the Thermotoga maritima C277Sperhydrolase (SEQ ID NO: 69) and assayed for an increase in the specificactivity for producing peroxycarboxylic acids from carboxylic acid estersubstrates. The assay conditions used to measure the respective specificactivities should be as close to identical as possible such that onlythe structure of the polypeptide having perhydrolytic activity varies.In one embodiment, reactions used to measure specific activity are runat ca. 25° C. in sodium bicarbonate buffer (10 mM, pH 6.5) containing 2mM triacetin, 10 mM hydrogen peroxide and approximately 2.0 μg/mL ofheat-treated extract supernatant total protein from E. coli strain KLP18expressing the C277T or C277S perhydrolase or variant perhydrolase,optionally in the presence of 2 mg/mL liquid laundry detergent(comprising 15-30% anionic and non-ionic surfactants, 5-15% soap, and<5% polycarboxylates, perfume, phosphonates, optical brighteners; seeExample 7).

In another embodiment, the reactions to measure specific activity areconducted under simulated laundry care conditions at ca. 20° C. using 0to 6 mg/mL liquid laundry detergent, 2 mM triacetin, 10 mM hydrogenperoxide (from sodium percarbonate), hard water plus percarbonate as abuffer (pH 10), and 2 μg/mL to 6 μg/mL of heat-treated extractsupernatant total soluble protein from E. coli strain KLP18 expressingthe T. maritima C277T or C277S perhydrolase or the present variantperhydrolase (see Example 8).

In another embodiment, the reactions to measure specific activity areconducted under simulated laundry care conditions at ca. 20° C. using 0to 6 mg/mL liquid laundry detergent, 1 mM triacetin, 4.4 mM hydrogenperoxide (from sodium percarbonate), hard water plus percarbonate as abuffer (pH 9), and 1 μg/mL to 6 μg/mL of heat-treated extractsupernatant total soluble protein from E. coli strain KLP18 expressingthe T. maritima C277T or C277S perhydrolase or the present variantperhydrolase (see Example 9).

In another embodiment, the reactions to measure specific activity areconducted at ca. 20° C. using 0 to 6 mg/mL liquid laundry detergent, 2mM 4-acetoxybenozoic acid (4ABA), 10 mM hydrogen peroxide (from sodiumpercarbonate), in sodium percarbonate buffer (pH 7), and 2 μg/mL to 6μg/mL of heat-treated extract supernatant total soluble protein from E.coli strain KLP18 expressing the T. maritima C277T or C277S perhydrolaseor the present variant perhydrolase (see Example 10).

In another embodiment, the reactions to measure specific activity areconducted under simulated laundry care conditions at ca. 25° C. usingabout 2 g/L solid laundry detergent, a formulation generally comprisinga mixture of surfactants (sodium dodecylbenzenesulfonate, C12-15Pareth-5, C12-15 Pareth-7, sodium stearate, and stearic acid), builders(sodium carbonate, zeolite, sodium silicate, and citric acid), binders(cellulose, PEG-75, dextrin, and sucrose), bulking agents (sodiumsulfate, sodium chloride, sodium bicarbonate, and calcium carbonate),structurants (sodium acrylic Acid/MA copolymer, and sodiumpolyacrylate), sequestrants (tetrasodium etidronate and calcium sodiumEDTMP), optical brighteners (disodiumanilinomorpholinotriazinylaminostilbenesulfonate), stabilizing agents,anti-redeposition agents, antifoaming agents, and softness extenders,0.5 mM triacetin, 2 mM H₂O₂ from sodium perborate in deionized water,and about 2.0 μg/mL of heat-treated extract supernatant total solubleprotein from E. coli strain KLP18 (as prepare in Example 6) expressingthe T. maritima C277T or C277S perhydrolase or the present variantperhydrolase (see Example 11).

Suitable Reaction Conditions for the Enzyme-Catalyzed Preparation ofPeroxycarboxylic Acids from Carboxylic Acid Esters and Hydrogen Peroxide

A process is provided to produce an aqueous formulation comprising atleast one peroxycarboxylic acid by reacting carboxylic acid esters andan inorganic peroxide (such as hydrogen peroxide, sodium perborate orsodium percarbonate) in the presence of an enzyme catalyst havingperhydrolysis activity, wherein the enzyme catalyst comprises, in oneembodiment, a polypeptide having an amino acid sequence selected fromthe group consisting of SEQ ID NOs: 12, 14, 16, 30, 34, 40, 46, and 102.In a further embodiment, the polypeptide has the amino acid sequence ofSEQ ID NO: 34.

In one embodiment, suitable substrates include one or more estersprovided by the following formula:

[X]_(m)R₅

-   -   wherein X=an ester group of the formula R₆C(O)O    -   R₆═C1 to C7 linear, branched or cyclic hydrocarbyl moiety,        optionally substituted with hydroxyl groups or C1 to C4 alkoxy        groups, wherein R₆ optionally comprises one or more ether        linkages for R₆═C2 to C7;    -   R₅=a C1 to C6 linear, branched, or cyclic hydrocarbyl moiety or        a five-membered cyclic heteroaromatic moiety or six-membered        cyclic aromatic or heteroaromatic moiety optionally substituted        with hydroxyl groups;    -   wherein each carbon atom in R₅ individually comprises no more        than one hydroxyl group or no more than one ester group or        carboxylic acid group;    -   wherein R₅ optionally comprises one or more ether linkages;    -   m=is an integer ranging from 1 to the number of carbon atoms in        R₅; and    -   wherein said esters have solubility in water of at least 5 ppm        at 25° C.

In another embodiment, R₆ is C1 to C7 linear hydrocarbyl moiety,optionally substituted with hydroxyl groups or C1 to C4 alkoxy groups,optionally comprising one or more ether linkages. In a further preferredembodiment, R₆ is C2 to C7 linear hydrocarbyl moiety, optionallysubstituted with hydroxyl groups, and/or optionally comprising one ormore ether linkages.

In one embodiment, the suitable substrate may include 2-acetoxybenzoicacid, 3-acetoxybenzoic acid, 4-acetoxybenzoic acid or mixtures thereof.

In another embodiment, suitable substrates also include one or moreglycerides of the formula:

wherein R₁═C1 to C21 straight chain or branched chain alkyl optionallysubstituted with an hydroxyl or a C1 to C4 alkoxy group and R₃ and R₄are individually H or R₁C(O). In one embodiment, the suitable substrateis a glyceride of the above formula wherein R₁═C1 to C7 straight chainor branched chain alkyl optionally substituted with an hydroxyl or a C1to C4 alkoxy group and R₃ and R₄ are individually H or R₁C(O).

In another aspect, suitable substrates may also include one or moreesters of the formula:

wherein R₁ is a C1 to C7 straight chain or branched chain alkyloptionally substituted with an hydroxyl or a C1 to C4 alkoxy group andR₂ is a C1 to C10 straight chain or branched chain alkyl, alkenyl,alkynyl, aryl, alkylaryl, alkylheteroaryl, heteroaryl, (CH₂CH₂O)_(n), or(CH₂CH(CH₃)—O)_(n)H and n is 1 to 10.

Suitable substrates may also include one or more acylated saccharidesselected from the group consisting of acylated mono-, di-, andpolysaccharides. In another embodiment, the acylated saccharides areselected from the group consisting of acetylated xylan, fragments ofacetylated xylan, acetylated xylose (such as xylose tetraacetate),acetylated glucose (such as α-D-glucose pentaacetate; β-D-glucosepentaacetate), β-D-galactose pentaacetate, sorbitol hexaacetate, sucroseoctaacetate, β-D-ribofuranose-1,2,3,5-tetraacetate,tri-O-acetyl-D-galactal, tri-O-acetyl-D-glucal, and acetylatedcellulose. In a preferred embodiment, the acetylated saccharide isselected from the group consisting ofβ-D-ribofuranose-1,2,3,5-tetraacetate, tri-O-acetyl-D-galactal,tri-O-acetyl-D-glucal, and acetylated cellulose.

In another embodiment, suitable substrates are selected from the groupconsisting of: monoacetin; diacetin; triacetin; monopropionin;dipropionin; tripropionin; monobutyrin; dibutyrin; tributyrin; glucosepentaacetate; xylose tetraacetate; acetylated xylan; acetylated xylanfragments; β-D-ribofuranose-1,2,3,5-tetraacetate;tri-O-acetyl-D-galactal; tri-O-acetyl-D-glucal; monoesters or diestersof 1,2-ethanediol, 1,2-propanediol, 1,3-propanediol, 1,2-butanediol,1,3-butanediol, 2,3-butanediol, 1,4-butanediol, 1,2-pentanediol,2,5-pentanediol, 1,6-pentanediol, 1,2-hexanediol, 2,5-hexanediol,1,6-hexanediol; and mixtures thereof.

In another embodiment, the carboxylic acid ester is selected from thegroup consisting of monoacetin, diacetin, triacetin, and combinationsthereof. In another embodiment, the substrate is a C1 to C6 polyolcomprising one or more ester groups. In a preferred embodiment, one ormore of the hydroxyl groups on the C1 to C6 polyol are substituted withone or more acetoxy groups (such as 1,3-propanediol diacetate,1,4-butanediol diacetate, etc.). In a further embodiment, the substrateis propylene glycol diacetate (PGDA), ethylene glycol diacetate (EGDA),or a mixture thereof.

In another embodiment, suitable substrates are selected from the groupconsisting of ethyl acetate; methyl lactate; ethyl lactate; methylglycolate; ethyl glycolate; methyl methoxyacetate; ethyl methoxyacetate;methyl 3-hydroxybutyrate; ethyl 3-hydroxybutyrate; triethyl 2-acetylcitrate; glucose pentaacetate; gluconolactone; glycerides (mono-, di-,and triglycerides) such as monoacetin, diacetin, triacetin,monopropionin, dipropionin (glyceryl dipropionate), tripropionin(1,2,3-tripropionylglycerol), monobutyrin, dibutyrin (glyceryldibutyrate), tributyrin (1,2,3-tributyrylglycerol); acetylatedsaccharides; and mixtures thereof.

In a further embodiment, suitable substrates are selected from the groupconsisting of monoacetin, diacetin, triacetin, monopropionin,dipropionin, tripropionin, monobutyrin, dibutyrin, tributyrin, ethylacetate, and ethyl lactate. In yet another aspect, the substrate isselected from the group consisting of diacetin, triacetin, ethylacetate, and ethyl lactate. In a most preferred embodiment, the suitablesubstrate comprises triacetin.

The carboxylic acid ester is present in the aqueous reaction formulationat a concentration sufficient to produce the desired concentration ofperoxycarboxylic acid upon enzyme-catalyzed perhydrolysis. Thecarboxylic acid ester need not be completely soluble in the aqueousreaction formulation, but has sufficient solubility to permit conversionof the ester by the perhydrolase catalyst to the correspondingperoxycarboxylic acid. The carboxylic acid ester is present in theaqueous reaction formulation at a concentration of 0.0005 wt % to 40 wt% of the aqueous reaction formulation, preferably at a concentration of0.01 wt % to 20 wt % of the aqueous reaction formulation, and morepreferably at a concentration of 0.05 wt % to 10 wt % of the aqueousreaction formulation. The wt % of carboxylic acid ester may optionallybe greater than the solubility limit of the carboxylic acid ester, suchthat the concentration of the carboxylic acid ester is at least 0.0005wt % in the aqueous reaction formulation that is comprised of water,enzyme catalyst, and source of peroxide, where the remainder of thecarboxylic acid ester remains as a second separate phase of a two-phaseaqueous/organic reaction formulation. Not all of the added carboxylicacid ester must immediately dissolve in the aqueous reactionformulation, and after an initial mixing of all reaction components,additional continuous or discontinuous mixing is optional.

The peroxycarboxylic acids produced by the present reaction componentsmay vary depending upon the selected substrates, so long as the presentenzyme catalyst is used. In one embodiment, the peroxycarboxylic acidproduced is peracetic acid, perpropionic acid, perbutyric acid,peroctanoic acid, perlactic acid, perglycolic acid, permethoxyaceticacid, per-β-hydroxybutyric acid, or mixtures thereof.

The peroxygen source may include, but is not limited to, hydrogenperoxide, hydrogen peroxide adducts (e.g., urea-hydrogen peroxide adduct(carbamide peroxide)), perborate salts and percarbonate salts.Alternatively, hydrogen peroxide can be generated in situ by thereaction of a substrate and oxygen catalyzed by an enzyme having oxidaseactivity (including, but not limited to, glucose oxidase, galactoseoxidase, sorbitol oxidase, hexose oxidase, alcohol oxidase, glyceroloxidase, monoamine oxidase, glycolate oxidase, lactate oxidase, pyruvateoxidase, oxalate oxidase, choline oxidase, cholesterol oxidase, pyranoseoxidase, carboxyalcohol oxidase, L-amino acid oxidase, glycine oxidase,glutamate oxidase, lysine oxidase, and uricase). The concentration ofperoxygen compound in the aqueous reaction formulation may range from0.0033 wt % to about 50 wt %, preferably from 0.033 wt % to about 40 wt%, more preferably from 0.33 wt % to about 30 wt %.

Many perhydrolase catalysts (such as whole cells, permeabilized wholecells, and partially purified whole cell extracts) have been reported tohave catalase activity (EC 1.11.1.6). Catalases catalyze the conversionof hydrogen peroxide into oxygen and water. In one aspect, the enzymecatalyst having perhydrolase activity lacks catalase activity. Inanother aspect, the enzyme catalyst having perhydrolase activity has asufficiently-low catalase activity that the presence of said catalaseactivity does not significantly interfere with perhydrolase-catalyzedperoxycarboxylic acid production. In another aspect, a catalaseinhibitor is added to the aqueous reaction formulation. Examples ofcatalase inhibitors include, but are not limited to, sodium azide andhydroxylamine sulfate. One of skill in the art can adjust theconcentration of catalase inhibitor as needed. The concentration of thecatalase inhibitor typically ranges from 0.1 mM to about 1 M; preferablyabout 1 mM to about 50 mM; more preferably from about 1 mM to about 20mM. In one aspect, sodium azide concentration typically ranges fromabout 20 mM to about 60 mM while hydroxylamine sulfate is concentrationis typically about 0.5 mM to about 30 mM, preferably about 10 mM.

The catalase activity in a host cell can be down-regulated or eliminatedby disrupting expression of the gene(s) responsible for the catalaseactivity using well known techniques including, but not limited to,transposon mutagenesis, RNA antisense expression, targeted mutagenesis,and random mutagenesis. In a preferred embodiment, the gene(s) encodingthe endogenous catalase activity are down-regulated or disrupted (i.e.,“knocked-out”). As used herein, a “disrupted” gene is one where theactivity and/or function of the protein encoded by the modified gene isno longer present. Means to disrupt a gene are well-known in the art andmay include, but are not limited to, insertions, deletions, or mutationsto the gene so long as the activity and/or function of the correspondingprotein is no longer present. In a further preferred embodiment, theproduction host is an E. coli production host comprising a disruptedcatalase gene selected from the group consisting of katG and katE (seeU.S. Pat. No. 7,951,566 to DiCosimo et al.). In another embodiment, theproduction host is an E. coli strain comprising a down-regulation and/ordisruption in both katG and katE catalase genes. An E. coli straincomprising a double-knockout of katG and katE has been prepared and isdescribed as E. coli strain KLP18 (U.S. Pat. No. 7,951,566 to DiCosimoet al.).

The concentration of the catalyst in the aqueous reaction formulationdepends on the specific catalytic activity of the catalyst, and ischosen to obtain the desired rate of reaction. The weight of catalyst inperhydrolysis reactions typically ranges from 0.0001 mg to 50 mg per mLof total reaction volume, preferably from 0.0005 mg to 10 mg per mL,more preferably from 0.0010 mg to 2.0 mg per mL. The catalyst may alsobe immobilized on a soluble or insoluble support using methodswell-known to those skilled in the art; see for example, Immobilizationof Enzymes and Cells; Gordon F. Bickerstaff, Editor; Humana Press,Totowa, N.J., USA; 1997. The use of immobilized catalysts permits therecovery and reuse of the catalyst in subsequent reactions. The enzymecatalyst may be in the form of whole microbial cells, permeabilizedmicrobial cells, microbial cell extracts, partially-purified or purifiedenzymes, and mixtures thereof.

In one aspect, the concentration of peroxycarboxylic acid generated bythe combination of chemical perhydrolysis and enzymatic perhydrolysis ofthe carboxylic acid ester is sufficient to provide an effectiveconcentration of peroxycarboxylic acid for disinfection, bleaching,sanitization, deodorizing or destaining at a desired pH. In anotheraspect, the peroxycarboxylic acid is generated at a safe and efficaciousconcentration suitable for use in a personal care product to be appliedto the hair, skin, nails or tissues of the oral cavity, such as toothenamel, tooth pellicle or the gums. In another aspect, the presentmethods provide combinations of enzymes and enzyme substrates to producethe desired effective concentration of peroxycarboxylic acid, where, inthe absence of added enzyme, there is a significantly lowerconcentration of peroxycarboxylic acid produced. Although there may besome chemical perhydrolysis of the enzyme substrate by direct chemicalreaction of inorganic peroxide with the enzyme substrate, there may notbe a sufficient concentration of peroxycarboxylic acid generated toprovide an effective concentration of peroxycarboxylic acid in thedesired applications, and a significant increase in totalperoxycarboxylic acid concentration is achieved by the addition of anappropriate perhydrolase catalyst to the aqueous reaction formulation.

In one aspect of the invention, the concentration of peroxycarboxylicacid generated (e.g. peracetic acid) by the enzymatic perhydrolysis isat least about 2 ppm, preferably at least 20 ppm, preferably at least100 ppm, more preferably at least about 200 ppm peroxycarboxylic acid,more preferably at least 300 ppm, more preferably at least 500 ppm, morepreferably at least 700 ppm, more preferably at least about 1000 ppmperoxycarboxylic acid, more preferably at least about 2000 ppmperoxycarboxylic acid, most preferably at least 10,000 ppmperoxycarboxylic acid within 5 minutes more preferably within 1 minuteof initiating the enzymatic perhydrolysis reaction. In a second aspectof the invention, the concentration of peroxycarboxylic acid generated(e.g. peracetic acid) by the enzymatic perhydrolysis is at least about 2ppm, preferably at least 20 ppm, preferably at least 30 ppm, morepreferably at least about 40 ppm peroxycarboxylic acid, more preferablyat least 50 ppm, more preferably at least 60 ppm, more preferably atleast 70 ppm, more preferably at least about 80 ppm peroxycarboxylicacid, most preferably at least 100 ppm peroxycarboxylic acid within 5minutes, more preferably within 1 minute, of initiating the enzymaticperhydrolysis reaction (i.e., time measured from combining the reactioncomponents to form the formulation).

The aqueous formulation comprising the peroxycarboxylic acid may beoptionally diluted with diluent comprising water, or a solutionpredominantly comprised of water, to produce a formulation with thedesired lower target concentration of peroxycarboxylic acid. In oneaspect, the reaction time required to produce the desired concentration(or concentration range) of peroxycarboxylic acid is about 20 minutes orless, preferable about 5 minutes or less, most preferably about 1 minuteor less.

In other aspects, the surface or inanimate object contaminated with aconcentration of a biological contaminant(s) is contacted with theperoxycarboxylic acid formed in accordance with the processes describedherein within about 1 minute to about 168 hours of combining saidreaction components, or within about 1 minute to about 48 hours, orwithin about 1 minute to 2 hours of combining said reaction components,or any such time interval therein.

In another aspect, the peroxycarboxylic acid formed in accordance withthe processes describe herein is used in a laundry care applicationwherein the peroxycarboxylic acid is contacted with clothing or atextile to provide a benefit, such as disinfecting, bleaching,destaining, deodorizing and/or a combination thereof. Theperoxycarboxylic acid may be used in a variety of laundry care productsincluding, but not limited to, laundry or textile pre-wash treatments,laundry detergents or additives, stain removers, bleaching compositions,deodorizing compositions, and rinsing agents. In one embodiment, thepresent process to produce a peroxycarboxylic acid for a target surfaceis conducted in situ.

In the context of laundry care applications, the term “contacting anarticle of clothing or textile” means that the article of clothing ortextile is exposed to a formulation disclosed herein. To this end, thereare a number of formats the formulation may be used to treat articles ofclothing or textiles including, but not limited to, liquid, solids, gel,paste, bars, tablets, spray, foam, powder, or granules and can bedelivered via hand dosing, unit dosing, dosing from a substrate,spraying and automatic dosing from a laundry washing or drying machine.Granular compositions can also be in compact form; liquid compositionscan also be in a concentrated form.

When the formulations disclosed herein are used in a laundry washingmachine, the formulation can further contain components typical tolaundry detergents. For example, typical components included, but arenot limited to, surfactants, bleaching agents, bleach activators,additional enzymes, suds suppressors, dispersants, lime-soapdispersants, soil suspension and anti-redeposition agents, softeningagents, corrosion inhibitors, tarnish inhibitors, germicides, pHadjusting agents, non-builder alkalinity sources, chelating agents,organic and/or inorganic fillers, solvents, hydrotropes, opticalbrighteners, dyes, and perfumes. For example, a liquid laundry detergentwas used in Example 6 comprising 15-30% anionic and non-ionicsurfactants, 5-15% soap, and <5% polycarboxylates, perfume,phosphonates, optical brighteners. In a further example, a solid laundrydetergent formulation was used in Example 11 generally comprisingsurfactants (sodium dodecylbenzenesulfonate, C12-15 Pareth-5, C12-15Pareth-7, sodium stearate, and stearic acid), builders (sodiumcarbonate, zeolite, sodium silicate, and citric acid), binders(cellulose, PEG-75, dextrin, and sucrose), bulking agents (sodiumsulfate, sodium chloride, sodium bicarbonate, and calcium carbonate),structurants (sodium acrylic Acid/MA copolymer, and sodiumpolyacrylate), sequestrants (tetrasodium etidronate and calcium sodiumEDTMP), optical brighteners (disodiumanilinomorpholinotriazinylaminostilbenesulfonate), stabilizing agents,anti-redeposition agents, antifoaming agents, and softness extenders.

The formulations disclosed herein can also be used as detergent additiveproducts in solid or liquid form. Such additive products are intended tosupplement or boost the performance of conventional detergentcompositions and can be added at any stage of the cleaning process.

In connection with the present systems and methods for laundry carewhere the peracid is generated for one or more of bleaching, stainremoval, and odor reduction, the concentration of peracid generated(e.g., peracetic acid) by the perhydrolysis of at least one carboxylicacid ester may be at least about 2 ppm, preferably at least 20 ppm,preferably at least 100 ppm, and more preferably at least about 200 ppmperacid. In connection with the present systems and methods for laundrycare where the peracid is generated for disinfection or sanitization,the concentration of peracid generated (e.g., peracetic acid) by theperhydrolysis of at least one carboxylic acid ester may be at leastabout 2 ppm, more preferably at least 20 ppm, more preferably at least200 ppm, more preferably at least 500 ppm, more preferably at least 700ppm, more preferably at least about 1000 ppm peracid, most preferably atleast 2000 ppm peracid within 10 minutes, preferably within 5 minutes,and most preferably within 1 minute of initiating the perhydrolysisreaction. The product formulation comprising the peracid may beoptionally diluted with water, or a solution predominantly comprised ofwater, to produce a formulation with the desired lower concentration ofperacid. In one aspect of the present methods and systems, the reactiontime required to produce the desired concentration of peracid is notgreater than about two hours, preferably not greater than about 30minutes, more preferably not greater than about 10 minutes, even morepreferably not greater than about 5 minutes, and most preferably inabout 1 minute or less.

The temperature of the reaction is chosen to control both the reactionrate and the stability of the enzyme catalyst activity. The temperatureof the reaction may range from just above the freezing point of theaqueous reaction formulation (approximately 0° C.) to about 85° C., witha preferred range of reaction temperature of from about 5° C. to about75° C.

The pH of the aqueous reaction formulation while enzymatically producingperoxycarboxylic acid is maintained at a pH ranging from about 5.0 toabout 10.0, preferably about 6.5 to about 8.5, and yet even morepreferably about 6.5 to about 7.5. In one embodiment, the pH of theaqueous reaction formulation ranges from about 6.5 to about 8.5 for atleast 30 minutes after combining the reaction components. The pH of theaqueous reaction formulation may be adjusted or controlled by theaddition or incorporation of a suitable buffer, including, but notlimited to, phosphate, pyrophosphate, bicarbonate, acetate, or citrate.In one embodiment, the buffer is selected from a phosphate buffer, abicarbonate buffer, or a buffer formed by the combination of hard ward(tap water to simulate laundry care applications) and percarbonate (fromsodium percarbonate used to generate hydrogen peroxide). Theconcentration of buffer, when employed, is typically from 0.1 mM to 1.0M, preferably from 1 mM to 300 mM, most preferably from 10 mM to 100 mM.In another aspect of the present invention, no buffer is added to thereaction mixture while enzymatically producing peroxycarboxylic acid.

In yet another aspect, the enzymatic perhydrolysis aqueous reactionformulation may contain an organic solvent that acts as a dispersant toenhance the rate of dissolution of the carboxylic acid ester in theaqueous reaction formulation. Such solvents include, but are not limitedto, propylene glycol methyl ether, acetone, cyclohexanone, diethyleneglycol butyl ether, tripropylene glycol methyl ether, diethylene glycolmethyl ether, propylene glycol butyl ether, dipropylene glycol methylether, cyclohexanol, benzyl alcohol, isopropanol, ethanol, propyleneglycol, and mixtures thereof.

In another aspect, the enzymatic perhydrolysis product may containadditional components that provide desirable functionality. Theseadditional components include, but are not limited to, buffers,detergent builders, thickening agents, emulsifiers, surfactants, wettingagents, corrosion inhibitors (e.g., benzotriazole), enzyme stabilizers,and peroxide stabilizers (e.g., metal ion chelating agents). Many of theadditional components are well known in the detergent industry (see, forexample, U.S. Pat. No. 5,932,532; hereby incorporated by reference).Examples of emulsifiers include, but are not limited to, polyvinylalcohol or polyvinylpyrrolidone. Examples of thickening agents include,but are not limited to, LAPONITE® RD, corn starch, PVP, CARBOWAX®,CARBOPOL®, CABOSIL®, polysorbate 20, PVA, and lecithin. Examples ofbuffering systems include, but are not limited to, sodium phosphatemonobasic/sodium phosphate dibasic; sulfamic acid/triethanolamine;citric acid/triethanolamine; tartaric acid/triethanolamine; succinicacid/triethanolamine; and acetic acid/triethanolamine. Examples ofsurfactants include, but are not limited to, a) non-ionic surfactantssuch as block copolymers of ethylene oxide or propylene oxide,ethoxylated or propoxylated linear and branched primary and secondaryalcohols, and aliphatic phosphine oxides b) cationic surfactants such asquaternary ammonium compounds, particularly quaternary ammoniumcompounds having a C8-C20 alkyl group bound to a nitrogen atomadditionally bound to three C1-C2 alkyl groups, c) anionic surfactantssuch as alkane carboxylic acids (e.g., C8-C20 fatty acids), alkylphosphonates, alkane sulfonates (e.g., sodium dodecylsulphate “SDS”) orlinear or branched alkyl benzene sulfonates, alkene sulfonates and d)amphoteric and zwitterionic surfactants such as aminocarboxylic acids,aminodicarboxylic acids, alkybetaines, and mixtures thereof. Additionalcomponents may include fragrances, dyes, stabilizers of hydrogenperoxide (e.g., metal chelators such as1-hydroxyethylidene-1,1-diphosphonic acid (DEQUEST® 2010, Solutia Inc.,St. Louis, Mo.) and ethylenediaminetetraacetic acid (EDTA)), TURPINAL®SL, DEQUEST® 0520, DEQUEST® 0531, stabilizers of enzyme activity (e.g.,polyethylene glycol (PEG)), and detergent builders.

In another aspect, the enzymatic perhydrolysis product may be pre-mixedto generate the desired concentration of peroxycarboxylic acid prior tocontacting the surface or inanimate object to be disinfected.

In another aspect, the enzymatic perhydrolysis product is not pre-mixedto generate the desired concentration of peroxycarboxylic acid prior tocontacting the surface or inanimate object to be disinfected, butinstead, the components of the aqueous reaction formulation thatgenerate the desired concentration of peroxycarboxylic acid arecontacted with the surface or inanimate object to be disinfected and/orbleached or destained, generating the desired concentration ofperoxycarboxylic acid. In some embodiments, the components of theaqueous reaction formulation combine or mix at the locus. In someembodiments, the reaction components are delivered or applied to thelocus and subsequently mix or combine to generate the desiredconcentration of peroxycarboxylic acid.

Production of Peroxycarboxylic Acids Using a Perhydrolase Catalyst

The peroxycarboxylic acids, once produced, are quite reactive and maydecrease in concentration over extended periods of time, depending onvariables that include, but are not limited to, temperature and pH. Assuch, it may be desirable to keep the various reaction componentsseparated, especially for liquid formulations. In one aspect, thehydrogen peroxide source is separate from either the substrate or theperhydrolase catalyst, preferably from both. This can be accomplishedusing a variety of techniques including, but not limited to, the use ofmulticompartment chambered dispensers (U.S. Pat. No. 4,585,150) and atthe time of use physically combining the perhydrolase catalyst with asource of peroxygen (such as hydrogen peroxide) and the presentsubstrates to initiate the aqueous enzymatic perhydrolysis reaction. Theperhydrolase catalyst may optionally be immobilized within the body ofreaction chamber or separated (e.g., filtered, etc.) from the reactionproduct comprising the peroxycarboxylic acid prior to contacting thesurface and/or object targeted for treatment. The perhydrolase catalystmay be in a liquid matrix or in a solid form (e.g., powder or tablet) orembedded within a solid matrix that is subsequently mixed with thesubstrates to initiate the enzymatic perhydrolysis reaction. In afurther aspect, the perhydrolase catalyst may be contained within adissolvable or porous pouch that may be added to the aqueous substratematrix to initiate enzymatic perhydrolysis. In yet a further aspect, theperhydrolase catalyst may comprise the contents contained within aseparate compartment of a dissolvable or porous pouch that has at leastone additional compartment for the containment contents comprising theester substrate and/or source of peroxide. In an additional furtheraspect, a powder comprising the enzyme catalyst is suspended in thesubstrate (e.g., triacetin), and at time of use is mixed with a sourceof peroxygen in water.

Method for Determining the Concentration of Peroxycarboxylic Acid andHydrogen Peroxide.

A variety of analytical methods can be used in the present method toanalyze the reactants and products including, but not limited to,titration, high performance liquid chromatography (HPLC), gaschromatography (GC), mass spectroscopy (MS), capillary electrophoresis(CE), the analytical procedure described by U. Karst et al., (Anal.Chem., 69(17):3623-3627 (1997)), and the2,2′-azino-bis(3-ethylbenzothazoline)-6-sulfonate (ABTS) assay (S.Minning, et al., Analytica Chimica Acta 378:293-298 (1999) and WO2004/058961 A1) as described in U.S. Pat. No. 7,951,566.

Determination of Minimum Biocidal Concentration of PeroxycarboxylicAcids

The method described by J. Gabrielson et al. (J. Microbiol. Methods 50:63-73 (2002)) can be employed for determination of the Minimum BiocidalConcentration (MBC) of peroxycarboxylic acids, or of hydrogen peroxideand enzyme substrates. The assay method is based on XTT reductioninhibition, where XTT(2,3-bis[2-methoxy-4-nitro-5-sulfophenyl]-5-[(phenylamino)carbonyl]-2H-tetrazolium,inner salt, monosodium salt) is a redox dye that indicates microbialrespiratory activity by a change in optical density (OD) measured at 490nm or 450 nm. However, there are a variety of other methods availablefor testing the activity of disinfectants and antiseptics including, butnot limited to, viable plate counts, direct microscopic counts, dryweight, turbidity measurements, absorbance, and bioluminescence (see,for example Brock, Semour S., Disinfection, Sterilization, andPreservation, 5^(th) edition, Lippincott Williams & Wilkins,Philadelphia, Pa., USA; 2001).

Uses of Enzymatically Prepared Peroxycarboxylic Acid Compositions

The enzyme catalyst-generated peroxycarboxylic acid produced accordingto the present method can be used in a variety of hard surface/inanimateobject applications for reduction of concentrations of biologicalcontaminants, such as decontamination of medical instruments (e.g.,endoscopes), textiles (such as garments and carpets), food preparationsurfaces, food storage and food-packaging equipment, materials used forthe packaging of food products, chicken hatcheries and grow-outfacilities, animal enclosures, and spent process waters that havemicrobial and/or virucidal activity. The enzyme-generatedperoxycarboxylic acids may be used in formulations designed toinactivate prions (e.g., certain proteases) to additionally providebiocidal activity (see U.S. Pat. No. 7,550,420 to DiCosimo et al.).

In one aspect, the peroxycarboxylic acid composition is useful as adisinfecting agent for non-autoclavable medical instruments and foodpackaging equipment. As the peroxycarboxylic acid-containing formulationmay be prepared using GRAS or food-grade components (enzyme, enzymesubstrate, hydrogen peroxide, and buffer), the enzyme-generatedperoxycarboxylic acid may also be used for decontamination of animalcarcasses, meat, fruits and vegetables, or for decontamination ofprepared foods. The enzyme-generated peroxycarboxylic acid may beincorporated into a product whose final form is a powder, liquid, gel,film, solid or aerosol. The enzyme-generated peroxycarboxylic acid maybe diluted to a concentration that still provides an efficaciousdecontamination.

The compositions comprising an efficacious concentration ofperoxycarboxylic acid can be used to disinfect surfaces and/or objectscontaminated (or suspected of being contaminated) with biologicalcontaminants, such as pathogenic microbial contaminants, by contactingthe surface or object with the products produced by the presentprocesses. As used herein, “contacting” refers to placing a disinfectingcomposition comprising an effective concentration of peroxycarboxylicacid in contact with the surface or inanimate object suspected ofcontamination with a biological contaminant for a period of timesufficient to clean and disinfect. Contacting includes spraying,treating, immersing, flushing, pouring on or in, mixing, combining,painting, coating, applying, affixing to and otherwise communicating aperoxycarboxylic acid solution or composition comprising an efficaciousconcentration of peroxycarboxylic acid, or a solution or compositionthat forms an efficacious concentration of peroxycarboxylic acid, withthe surface or inanimate object suspected of being contaminated with aconcentration of a biological contaminant. The disinfectant compositionsmay be combined with a cleaning composition to provide both cleaning anddisinfection. Alternatively, a cleaning agent (e.g., a surfactant ordetergent) may be incorporated into the formulation to provide bothcleaning and disinfection in a single composition.

The compositions comprising an efficacious concentration ofperoxycarboxylic add can also contain at least one additionalantimicrobial agent, combinations of prion-degrading proteases, avirucide, a sporicide, or a biocide. Combinations of these agents withthe peroxycarboxylic acid produced by the claimed processes can providefor increased and/or synergistic effects when used to clean anddisinfect surfaces and/or objects contaminated (or suspected of beingcontaminated) with biological contaminants. Suitable antimicrobialagents include carboxylic esters (e.g., p-hydroxy alkyl benzoates andalkyl cinnamates), sulfonic acids (e.g., dodecylbenzene sulfonic acid),iodo-compounds or active halogen compounds (e.g., elemental halogens,halogen oxides (e.g., NaOCl, HOCl, HOBr, ClO₂), iodine, interhalides(e.g., iodine monochloride, iodine dichloride, iodine trichloride,iodine tetrachloride, bromine chloride, iodine monobromide, or iodinedibromide), polyhalides, hypochlorite salts, hypochlorous acid,hypobromite salts, hypobromous acid, chloro- and bromo-hydantoins,chlorine dioxide, and sodium chlorite), organic peroxides includingbenzoyl peroxide, alkyl benzoyl peroxides, ozone, singlet oxygengenerators, and mixtures thereof, phenolic derivatives (e.g., o-phenylphenol, o-benzyl-p-chlorophenol, tert-amyl phenol and C₁-C₆ alkylhydroxy benzoates), quaternary ammonium compounds (e.g.,alkyldimethylbenzyl ammonium chloride, dialkyldimethyl ammonium chlorideand mixtures thereof), and mixtures of such antimicrobial agents, in anamount sufficient to provide the desired degree of microbial protection.Effective amounts of antimicrobial agents include about 0.001 wt % toabout 60 wt % antimicrobial agent, about 0.01 wt % to about 15 wt %antimicrobial agent, or about 0.08 wt % to about 2.5 wt % antimicrobialagent.

In one aspect, the peroxycarboxylic acids formed by the process can beused to reduce the concentration of viable biological contaminants (suchas a microbial population) when applied on and/or at a locus. As usedherein, a “locus” comprises part or all of a target surface suitable fordisinfecting or bleaching. Target surfaces include all surfaces that canpotentially be contaminated with biological contaminants. Non-limitingexamples include equipment surfaces found in the food or beverageindustry (such as tanks, conveyors, floors, drains, coolers, freezers,equipment surfaces, walls, valves, belts, pipes, drains, joints,crevasses, combinations thereof, and the like); building surfaces (suchas walls, floors and windows); non-food-industry related pipes anddrains, including water treatment facilities, pools and spas, andfermentation tanks; hospital or veterinary surfaces (such as walls,floors, beds, equipment (such as endoscopes), clothing worn inhospital/veterinary or other healthcare settings, including clothing,scrubs, shoes, and other hospital or veterinary surfaces); restaurantsurfaces; bathroom surfaces; toilets; clothes and shoes; surfaces ofbarns or stables for livestock, such as poultry, cattle, dairy cows,goats, horses and pigs; hatcheries for poultry or for shrimp; andpharmaceutical or biopharmaceutical surfaces (e.g., pharmaceutical orbiopharmaceutical manufacturing equipment, pharmaceutical orbiopharmaceutical ingredients, pharmaceutical or biopharmaceuticalexcipients). Additional hard surfaces include food products, such asbeef, poultry, pork, vegetables, fruits, seafood, combinations thereof,and the like. The locus can also include water absorbent materials suchas infected linens or other textiles. The locus also includes harvestedplants or plant products including seeds, corms, tubers, fruit, andvegetables, growing plants, and especially crop growing plants,including cereals, leaf vegetables and salad crops, root vegetables,legumes, berried fruits, citrus fruits and hard fruits.

Non-limiting examples of hard surface materials are metals (e.g., steel,stainless steel, chrome, titanium, iron, copper, brass, aluminum, andalloys thereof), minerals (e.g., concrete), polymers and plastics (e.g.,polyolefins, such as polyethylene, polypropylene, polystyrene,poly(meth)acrylate, polyacrylonitrile, polybutadiene,poly(acrylonitrile, butadiene, styrene), poly(acrylonitrile, butadiene),acrylonitrile butadiene; polyesters such as polyethylene terephthalate;and polyamides such as nylon). Additional surfaces include brick, tile,ceramic, porcelain, wood, wood pulp, paper, vinyl, linoleum, and carpet.

The peroxycarboxylic acids formed by the present process may be used toprovide a benefit to an article of clothing or a textile including, butnot limited to disinfecting, sanitizing, bleaching, destaining, anddeodorizing. The peroxycarboxylic acids formed by the present processmay be used in any number of laundry care products including, but notlimited to textile pre-wash treatments, laundry detergents, laundrydetergents or additives, stain removers, bleaching compositions,deodorizing compositions, and rinsing agents, to name a few.

The peroxycarboxylic acids formed by the present process can be used inone or more steps of the wood pulp or paper pulpbleaching/delignification process, particularly where peracetic acid isused (for example, see EP1040222 B1 and U.S. Pat. No. 5,552,018 toDevenyns, J.).

Recombinant Microbial Expression

The genes and gene products of the instant sequences may be produced inheterologous host cells, particularly in the cells of microbial hosts.Preferred heterologous host cells for expression of the instant genesand nucleic acid molecules are microbial hosts that can be found withinthe fungal or bacterial families and which grow over a wide range oftemperature, pH values, and solvent tolerances. For example, it iscontemplated that any of bacteria, yeast, and filamentous fungi maysuitably host the expression of the present nucleic acid molecules. Theperhydrolase may be expressed intracellularly, extracellularly, or acombination of both intracellularly and extracellularly, whereextracellular expression renders recovery of the desired protein from afermentation product more facile than methods for recovery of proteinproduced by intracellular expression. Transcription, translation and theprotein biosynthetic apparatus remain invariant relative to the cellularfeedstock used to generate cellular biomass; functional genes will beexpressed regardless. Examples of host strains include, but are notlimited to, bacterial, fungal or yeast species such as Aspergillus,Trichoderma, Saccharomyces, Pichia, Pharma, Kluyveromyces, Candida,Hansenula, Yarrowia, Salmonella, Bacillus, Acinetobacter, Zymomonas,Agrobacterium, Erythrobacter, Chlorobium, Chromatium, Flavobacterium,Cytophaga, Rhodobacter, Rhodococcus, Streptomyces, Brevibacterium,Corynebacteria, Mycobacterium, Deinococcus, Escherichia, Erwinia,Pantoea, Pseudomonas, Sphingomonas, Methylomonas, Methylobacter,Methylococcus, Methylosinus, Methylomicrobium, Methylocystis,Alcaligenes, Synechocystis, Synechococcus, Anabaena, Thiobacillus,Methanobacterium, Klebsiella, and Myxococcus. In one embodiment,bacterial host strains include Escherichia, Bacillus, and Pseudomonas.In a preferred embodiment, the bacterial host cell is Escherichia coli.

INDUSTRIAL PRODUCTION

A variety of culture methodologies may be applied to produce theperhydrolase catalyst. Large-scale production of a specific gene productover expressed from a recombinant microbial host may be produced bybatch, fed-batch or continuous culture methodologies. Batch andfed-batch culturing methods are common and well known in the art andexamples may be found in Thomas D. Brock in Biotechnology: A Textbook ofIndustrial Microbiology, Second Edition, Sinauer Associates, Inc.,Sunderland, Mass. (1989) and Deshpande, Mukund V., App. Biochem,Biotechnol., 36:227 (1992).

In one embodiment, commercial production of the desired perhydrolasecatalyst is accomplished with a continuous culture. Continuous culturesare an open system where a defined culture media is added continuouslyto a bioreactor and an equal amount of conditioned media is removedsimultaneously for processing. Continuous cultures generally maintainthe cells at a constant high liquid phase density where cells areprimarily in log phase growth. Alternatively, continuous culture may bepracticed with immobilized cells where carbon and nutrients arecontinuously added and valuable products, by-products or waste productsare continuously removed from the cell mass. Cell immobilization may beperformed using a wide range of solid supports composed of naturaland/or synthetic materials.

Recovery of the desired perhydrolase catalysts from a batch or fed-batchfermentation, or continuous culture may be accomplished by any of themethods that are known to those skilled in the art. For example, whenthe enzyme catalyst is produced intracellularly, the cell paste isseparated from the culture medium by centrifugation or membranefiltration, optionally washed with water or an aqueous buffer at adesired pH, then a suspension of the cell paste in an aqueous buffer ata desired pH is homogenized to produce a cell extract containing thedesired enzyme catalyst. The cell extract may optionally be filteredthrough an appropriate filter aid such as celite or silica to removecell debris prior to a heat-treatment step to precipitate undesiredprotein from the enzyme catalyst solution. The solution containing thedesired enzyme catalyst may then be separated from the precipitated celldebris and protein produced during the heat-treatment step by membranefiltration or centrifugation, and the resulting partially-purifiedenzyme catalyst solution concentrated by additional membrane filtration,then optionally mixed with an appropriate excipient (for example,maltodextrin, trehalose, sucrose, lactose, sorbitol, mannitol, phosphatebuffer, citrate buffer, or mixtures thereof) and spray-dried to producea solid powder comprising the desired enzyme catalyst. Alternatively,the resulting partially-purified enzyme catalyst solution prepared asdescribed above may be optionally concentrated by additional membranefiltration, and the partially-purified enzyme catalyst solution orresulting enzyme concentrate is then optionally mixed with one or morestabilizing agents (e.g., glycerol, sorbitol, propylene glycol,1,3-propanediol, polyols, polymeric polyols, polyvinylalcohol ormixtures thereof), one or more salts (e.g., sodium chloride, sodiumsulfate, potassium chloride, potassium sulfate, or mixtures thereof),and one or more biocides, and maintained as an aqueous solution untilused.

When an amount, concentration, or other value or parameter is giveneither as a range, preferred range, or a list of upper preferable valuesand lower preferable values, this is to be understood as specificallydisclosing all ranges formed from any pair of any upper range limit orpreferred value and any lower range limit or preferred value, regardlessof whether ranges are separately disclosed. Where a range of numericalvalues is recited herein, unless otherwise stated, the range is intendedto include the endpoints thereof, and all integers and fractions withinthe range. It is not intended that the scope be limited to the specificvalues recited when defining a range.

General Methods

The following examples are provided to demonstrate differentembodiments. It should be appreciated by those of skill in the art thatthe techniques disclosed in the examples which follow representtechniques discovered by the inventor to function well in the practiceof the methods disclosed herein, and thus can be considered toconstitute preferred modes for its practice. However, those of skill inthe art should, in light of the present disclosure, appreciate that manychanges can be made in the specific embodiments which are disclosed andstill obtain a like or similar result without departing from the spiritand scope of the presently disclosed methods.

All reagents and materials were obtained from DIFCO Laboratories(Detroit, Mich.), GIBCO/BRL (Gaithersburg, Md.), TCI America (Portland,Oreg.), Roche Diagnostics Corporation (Indianapolis, Ind.) orSigma-Aldrich Chemical Company (St. Louis, Mo.), unless otherwisespecified.

The following abbreviations in the specification correspond to units ofmeasure, techniques, properties, or compounds as follows: “sec” or “s”means second(s), “min” means minute(s), “h” or “hr” means hour(s), “μL”means microliter(s), “mL” means milliliter(s), “L” means liter(s), “mM”means millimolar, “M” means molar, “mmol” means millimole(s), “ppm”means part(s) per million, “wt” means weight, “wt %” means weightpercent, “g” means gram(s), “μg” means microgram(s), “ng” meansnanogram(s), “g” means gravity, “HPLC” means high performance liquidchromatography, “dd H₂O” means distilled and deionized water, “dcw”means dry cell weight, “ATCC” or “ATCC®” means the American Type CultureCollection (Manassas, Va.), “U” means unit(s) of perhydrolase activity,“rpm” means revolution(s) per minute, and “EDTA” meansethylenediaminetetraacetic acid.

Example 1 Construction of a Random Mutagenesis Library of Thermotogamaritima Acetyl Xylan Esterase C277T Variant

The coding sequence of a Thermotoga maritima acetyl xylan esterase(GENBANK® accession # NP_(—)227893.1) was synthesized using codonsoptimized for expression in E. coli (DNA 2.0, Menlo Park, Calif.) andcloned into pUC19 between Pst1 and Xba1 to create the plasmid known aspSW202 (U.S. Patent Application Publication No 2008-0176299). Thecodon-optimized sequence is provided as SEQ ID NO: 1 encoding thewild-type Thermotoga maritima acetyl xylan esterase provided as SEQ IDNO: 2.

A codon change was made in the gene using primer pairs identified as SEQID NO: 3 and SEQ ID NO: 4, and the QUIKCHANGE® site-directed mutagenesiskit (Stratagene, La Jolla, Calif.), according to the manufacturer'sinstructions, resulting in the amino acid change C277T (SEQ ID NO: 5),to create the plasmid known as pSW202/C277T (SEQ ID NO: 6). PlasmidpSW202/C277T served as a template for error-prone PCR using primersidentified as SEQ ID NO: 7 and SEQ ID NO: 8, and the GENEMORPH® IIrandom mutagenesis kit (Stratagene), according to the manufacturer'srecommendations. The resulting PCR product was digested with Pst1 andXba1 and ligated with pUC19 also digested with Pst1 and Xba1. E. coliKLP18 (see U.S. Patent Application Publication No. 2008-0176299) wastransformed with the ligation mixture and plated onto LB platessupplemented with 0.1 mg ampicillin/mL. Nucleotide sequencing of arandom sample indicated a mutation frequency of 2-8 changes per PCRproduct.

Example 2 Screening of Thermotoga maritima Error-Prone PCR Library forIncreased Enzyme Activity

Colonies were picked (automated) and placed into 96-well “master plates”containing 0.1 mL LB media supplemented with 0.1 mg ampicillin/mL andgrown 16-18 h at 37° C. and 80% humidity. From each well of the masterplates, 0.003 mL of culture was transferred to 96-well “inductionplates” containing 0.3 mL LB media supplemented with 0.1 mgampicillin/mL and 0.5 mM IPTG, which were incubated for 16-18 h withshaking at 37° C. and 80% humidity. Separately, 0.1 mL of 50% glycerolwas added to each well of the master plates, which were stored at −80°C. as stocks. From each well of the induction plates, 0.01 mL of culturewas transferred to 96-well “lysis plates” containing 0.09 mL of 56 mg/mLCELLYTIC™ Express (Sigma Aldrich, St. Louis, Mo.), which were incubatedfor 30 minutes at 30° C. From each well of the lysis plates, 0.01 mL ofmaterial was transferred to 96-well “assay plates” containing 0.045 mL“assay solution part 1” (10 mM triacetin, 50 mM potassium phosphatebuffer, pH 7.0). To each well of the assay plates was then added 0.045mL of “assay solution part 2” (20 mM hydrogen peroxide). Plates weregently mixed and incubated for 2 minutes at 30° C. To each well of theassay plate was added 0.1 mL of “stop buffer” (100 mM o-phenylenediamineand 500 mM sodium dihydrogen phosphate, pH 2.0). The plates wereincubated for 30 minutes at 30° C., after which absorbance at 458 nm wasread. T. maritima WT (codon optimized gene (SEQ ID NO: 1) encoding thewild type enzyme (SEQ ID NO: 2)) and T. maritima C277T (SEQ ID NO: 5)were incorporated into each plate as controls. Screening approximately7000 colonies resulted in the identification of numerous “hits”demonstrating activity significantly greater than T. maritima C277T (SEQID NO: 5). Nucleotide sequencing was used to determine the amino acidchanges in the perhydrolase enzyme of these hits (Table 1).

TABLE 1 T. maritima perhydrolase variants having perhydrolytic activitygreater than variant C277T identified in primary screen. # of nucleicamino amino acid acid variant acid SEQ SEQ ID changes amino acid changesrelative to T. maritima WT sequence ID ID 006D11 5 W28R P84S S201G E243DC277T 9 10 006F12 3 R261S I264F C277T 11 12 006D10 4 W28C L32P D151EC277T 13 14 006B12 3 W28C F104S C277T 15 16 006E11 2 V183D C277T 17 18006H5 2 C277T L318P 19 20 006B5 3 P265L C277T I297N 21 22 006F3 6 P16LF38S T53N R159C V183D C277T 23 24 006G10 4 R15H Q176H L208Q C277T 25 26006F1 2 L32P C277T 27 28 006A10 2 F268S C277T 29 30 006G6 4 E29G S62PF174L C277T 31 32 006E10 3 R218C C277T F317L 33 34 006D9 2 C277T R296P35 36 006F7 3 E9K G255C C277T 37 38 006E12 4 H227L T233A C277T A290V 3940 006F8 3 Q67R K82E C277T 41 42 006G8 3 F247L C277T V282D 43 44 006G112 D254G C277T 45 46 006E9 3 E17V R261H C277T 47 48 006F10 5 F24L T31SE48V C277T K316E 49 50 006F5 5 V43G G63C H100L C277T A284V 51 52 006E7 4F258Y E294G C277T L318P 53 54 006G12 2 F161L C277T 55 56 006H01 2 F253SC277T 57 58 006G2 2 F24I C277T 59 60 006F6 2 A206T C277T 61 62 006C10 2E55K C277T 63 64 006F9 2 Q67R C277T 65 66 control 1 C277T 5

Example 3 Construction of Site Saturation Libraries of Thermotogamaritima Acetyl Xylan Esterase C277S Variants

Using the codon-optimized Thermotoga maritima gene encoding acetyl xylanesterase described in Example 1 (SEQ ID NO: 1 encoding SEQ ID NO: 2), acodon change was made in the gene using primer pairs identified as SEQID NO: 67 and SEQ ID NO: 68, and the QUIKCHANGE® site-directedmutagenesis kit (Stratagene, La Jolla, Calif.), according to themanufacturer's instructions, resulting in a gene containing the aminoacid change C277S (SEQ ID NO: 69), to create the plasmid known aspSW202/C277S (SEQ ID NO: 70). Based on previous screening results ofrandom mutagenesis libraries, 6 residues (535, K77, L125, A266, N275 andN₃O₂) were selected for saturation mutagenesis. Six site saturationmini-libraries (approx 500 members each) were constructed usingdegenerate primers pairs (SEQ ID NO: 71-82) and the QUIKCHANGE®site-directed mutagenesis kit (Stratagene, La Jolla, Calif.).

Example 4 Screening of Thermotoga maritima Site Saturation MutagenesisLibrary for Increased Enzyme Activity

Colonies were picked (automated) and placed into 96-well “master plates”containing 0.1 mL LB media supplemented with 0.1 mg ampicillin/mL andgrown 16-18 h at 37° C. and 80% humidity. From each well of the masterplates, 0.003 mL of culture was transferred to 96-well “inductionplates” containing 0.3 mL LB media supplemented with 0.1 mgampicillin/mL and 0.5 mM IPTG, which were incubated for 16-18 h withshaking at 37° C. and 80% humidity. Separately, 0.1 mL of 50% glycerolwas added to each well of the master plates, which were stored at −80°C. as stocks. From each well of the induction plates, 0.01 mL of culturewas transferred to 96-well “lysis plates” containing 0.09 mL of 56 mg/mLCELLYTIC™ Express (Sigma Aldrich, St. Louis, Mo.), which were incubatedfor 30 minutes at 30° C. From each well of the lysis plates, 0.01 mL ofmaterial was transferred to 96-well “assay plates” containing 0.045 mL“assay solution part 1” (10 mM triacetin, 50 mM potassium phosphatebuffer, pH TO). To each well of the assay plates was then added 0.045 mLof “assay solution part 2” (20 mM hydrogen peroxide). Plates were gentlymixed and incubated for 2 minutes at 30° C. To each well of the assayplate was added 0.1 mL of “stop buffer” (100 mM o-phenylenediamine and500 mM sodium dihydrogen phosphate, pH 2.0). The plates were incubatedfor 30 minutes at 30° C., after which absorbance at 458 nm was read. T.maritima WT (codon optimized gene (SEQ ID NO: 1) encoding the wild typeenzyme (SEQ ID NO: 2)) and T. maritima C277S (SEQ ID NO: 69) wereincorporated into each plate as controls. Screening approximately 3000colonies resulted in the identification of numerous “hits” demonstratingactivity significantly greater than T. maritima C277S (SEQ ID NO: 69).Nucleotide sequencing was used to determine the amino acid changes inthe perhydrolase enzyme of these hits (Table 2).

TABLE 2 T. maritima perhydrolase variants having perhydrolytic activitygreater than variant C277S. mutations nucleic amino relative acid acidID to WT SEQ ID SEQ ID 873C8 S35R C277S 83 84 873D7 S35K C277S 85 86873D3 S35E C277S 87 88 873C9 S35N C277S 89 90 873H5 K77A C277S 91 92873G12 K77V C277S 93 94 873H1 K77D C277S 95 96 873A4 L125M C277S 97 98873A9 L125R C277S 99 100 873B4 A266P C277S 101 102 873B2 A266D C277S 103104 873G7 A266I C277S 105 106 873C3 A266E C277S 107 108 873B3 A266NC277S 109 110 873B12 A266R C277S 111 112 873G3 A266Q C277S 113 114 873E2A266V C277S 115 116

Example 5 Production of Variant Thermotoga maritima Perhydrolases

Variant strains identified in screens were grown in LB media at 37° C.with shaking up to OD_(600 nm)=0.4-0.5, at which time IPTG was added toa final concentration of 1 mM, and incubation continued for 2-3 h. Cellswere harvested by centrifugation and SDS-PAGE was performed to confirmexpression of the perhydrolase enzyme at 20-40% of total solubleprotein.

Example 6 Preparation of Heat-Treated Cell Extracts ContainingSemi-Purified Variant Thermotoga maritima Perhydrolases

Cell cultures (prepared as described in Example 5) were harvested bycentrifugation at 5,000×g for 15 minutes then resuspended (20% w/v) in50 mM phosphate buffer pH 7.0 supplemented with 1.0 mM DTT. Resuspendedcells were passed through a French pressure cell twice to ensure >95%cell lysis. Lysed cells were centrifuged for 30 minutes at 12,000×g, andthe supernatant was heated at 75° C. for 20 minutes, followed byquenching in an ice bath for 2 minutes. Precipitated protein was removedby centrifugation for 10 minutes at 11,000×g. The resulting heat-treatedextract supernatants were analyzed for mg total soluble protein/mL usingBCA assay, and stored frozen at −80° C. Analysis of a first set ofheat-treated extract supernatants produced by this method using PAGEindicated that the perhydrolase variant comprised approximately 85-90%of the total soluble protein in the heat-treated extract supernatant.Analysis of a second set of heat-treated extract supernatants producedby this method using PAGE indicated that the perhydrolase variantcomprised approximately 65-70% of the total soluble protein in theheat-treated extract supernatant. In the following Examples, theindividual Tables contain data obtained using a single set ofheat-treated extract supernatants that contained the same concentrationof perhydrolase variant as a percentage of total soluble protein.

Example 7 Comparison of Perhydrolase Variants Vs. Thermotoga maritimaC277T Variant Perhydrolase Using 2 mM Triacetin and 10 mM H₂O₂ in SodiumBicarbonate Buffer (10 mM, pH 6.5) at 20° C.

Reactions (100 mL total volume) were run at 20° C. in sodium bicarbonatebuffer (10 mM, pH 6.5) containing triacetin (2 mM), hydrogen peroxide(10 mM) and 2.0 μg/mL of heat-treated extract total soluble proteincontaining an error-prone PCR (epPCR)-generated variant perhydrolaseprepared as described in Example 6. Reactions were stirred for only thefirst 45 seconds of reaction to initially mix the reactants and enzyme.

A comparative reaction was also run under identical conditions to thatdescribed immediately above using 2.0 μg/mL of heat-treated extracttotal soluble protein isolated from E. coli KLP18/pSW228/C277T(expressing Thermotoga maritima C277T perhydrolase variant (SEQ ID NO:5)), where the heat-treated extract supernatant was prepared accordingto the procedure of Example 6, and where this heat-treated extractsupernatant contained the same concentration of Thermotoga maritimaC277T perhydrolase variant (SEQ ID NO: 5) as a percentage of totalsoluble protein as was present in the heat-treated extract total solubleprotein containing an error-prone PCR (epPCR)-generated variantperhydrolase.

Reaction samples were analyzed for the amount of peracetic acid (PAA)produced at 5 minutes and 20 minutes after combining the reactioncomponents using a modification of the method described by Karst et al.,supra. A sample (0.200 mL) of the reaction mixture was removed at apredetermined time and immediately mixed with 0.200 mL of 50 mMphosphoric acid in water to terminate the reaction by adjusting the pHof the diluted sample to less than pH 4. The resulting solution wasfiltered using an ULTRAFREE® MC-filter unit (30,000 Normal MolecularWeight Limit (NMWL), Millipore Corp., Billerica, Mass.; cat #UFC3LKT 00)by centrifugation for 2 min at 12,000 rpm. An aliquot (0.100 mL) of theresulting filtrate was transferred to a 1.5-mL screw cap HPLC vial(Agilent Technologies, Palo Alto, Calif.; #5182-0715) containing 0.300mL of deionized water, then 0.100 mL of 20 mM MTS (methyl-p-tolylsulfide) in acetonitrile was added, the vial capped, and the contentsbriefly mixed prior to a 10 min incubation at ca. 25° C. in the absenceof light. To the vial was then added 0.400 mL of acetonitrile and 0.100mL of a solution of triphenylphosphine (TPP, 120 mM) in acetonitrile,the vial re-capped, and the resulting solution mixed and incubated atca. 25° C. for 30 min in the absence of light. To the vial was thenadded 0.100 mL of 2.5 mM N,N-diethyl-m-toluamide (DEET; HPLC externalstandard) and the resulting solution analyzed by HPLC for MTSO(methyl-p-tolyl sulfoxide), the stoichiometric oxidation productproduced by reaction of MTS with peracetic acid. A control reaction wasrun in the absence of added extract protein or triacetin to determinethe rate of oxidation of MTS in the assay mixture by hydrogen peroxide,for correction of the rate of peracetic acid production for backgroundMTS oxidation. HPLC method: Supelco Discovery C8 column (10-cm×4.0-mm, 5μm) (catalog #569422-U) with Supelco Supelguard Discovery C8 precolumn(Sigma-Aldrich; catalog #59590-U); 10 microliter injection volume;gradient method with CH₃CN (Sigma-Aldrich; catalog #270717) anddeionized water at 1.0 mL/min and ambient temperature (Table 3).

TABLE 3 HPLC Gradient for analysis of peracetic acid. Time (min:sec) (%CH₃CN) 0:00 40 3:00 40 3:10 100 4:00 100 4:10 40 7:00 (stop) 40

Several variants were identified that produced a higher concentration ofPAA at one or both time points when compared to the T. maritima C277Tvariant perhydrolase (Published U.S. Patent Application No. 2010-0087529to DiCosimo et al., SEQ ID NO: 5). As shown in Tables 4 and 5, variants006B12 (W28C/F104S/C277T; SEQ ID NO: 16), 006F12 (R261S/I264F/C277T; SEQID NO: 12), and 006D10 (W28C/L32P/D151E/C277T; SEQ ID NO: 14) were ableto produce more peracetic acid when compared to the T. maritima C277Tperhydrolase (SEQ ID NO: 5) under the specified conditions (either withor without added liquid detergent at pH 6.5).

TABLE 4 Peracetic acid (PAA) production at 5 minutes and 20 minutes fromvariants 006B12 vs. T. maritima C277T in sodium bicarbonate buffer (10mM, pH 6.5) at 20° C., 2.0 μg/mL heat-treated extract total solubleprotein and 0 mg/mL and 2.0 mg/mL liquid laundry detergent. Totalsoluble Liquid PAA @ PAA @ SEQ ID Triacetin H₂O₂ protein detergent 5min. 20 min. Variant ID NO: Samples (mM) (mM) (μg/mL) (mg/mL) Initial pH(ppm) (ppm) no perhydrolase 2 10 0 0 6.5 0.2 1.4 Control 5 C277T 2 102.0 0 6.5 51 73 Control 5 C277T 2 10 2.0 0 6.5 46 68 006B12 16W28C/F104S/C277T 2 10 2.0 0 6.5 48 63 006B12 16 W28C/F104S/C277T 2 102.0 0 6.5 53 71 no perhydrolase 2 10 0 2 6.5 0.7 0.7 Control 5 C277T 210 2.0 2 6.5 26 45 Control 5 C277T 2 10 2.0 2 6.5 24 44 006B12 16W28C/F104S/C277T 2 10 2.0 2 6.5 32 48 006B12 16 W28C/F104S/C277T 2 102.0 2 6.5 36 50

TABLE 5 Peracetic acid (PAA) production at 5 minutes and 20 minutes fromvariants 006F12 and 006D10 vs. T. maritima C277T in sodium bicarbonatebuffer (10 mM, pH 7.2) at 20° C., 2.0 μg/mL heat-treated extract totalsoluble protein, and 0 mg/mL or 2.0 mg/mL liquid laundry detergent.Total soluble Liquid PAA @ PAA @ Variant SEQ ID Triacetin H₂O₂ proteindetergent Initial 5 min. 20 min. ID NO: Samples (mM) (mM) (μg/mL)(mg/mL) pH (ppm) (ppm) no perhydrolase 2 10 0 0 10 0.2 1.4 Control 5C277T 2 10 2.0 0 10 39 70 Control 5 C277T 2 10 2.0 0 10 38 63 006F12 12R261S/I264F/C277T 2 10 2.0 0 10 51 75 006F12 12 R261S/I264F/C277T 2 102.0 0 10 49 68 006D10 14 W28C/L32P/D151E/C277T 2 10 2.0 0 10 45 69006D10 14 W28C/L32P/D151E/C277T 2 10 2.0 0 10 44 61 no perhydrolase 2 100 2 10 0.7 0.7 Control 5 C277T 2 10 2.0 2 10 26 45 Control 5 C277T 2 102.0 2 10 22 39 006F12 12 R261S/I264F/C277T 2 10 2.0 2 10 31 51 006F12 12R261S/I264F/C277T 2 10 2.0 2 10 27 44 006D10 14 W28C/L32P/D151E/C277T 210 2.0 2 10 26 39 006D10 14 W28C/L32P/D151E/C277T 2 10 2.0 2 10 25 38

Example 8 Comparison of Thermotoga maritima Perhydrolase Variants Vs.Thermotoga maritima C277T Perhydrolase Under Simulated Laundry CareApplication Conditions

Five error-prone PCR (epPCR)-generated variant perhydrolases wereevaluated for peracetic acid (PAA) production under laundry applicationconditions (10 mL total volume, 2 mM triacetin (TA), 10 mM H₂O₂ fromsodium percarbonate in hard water (HW, 400 ppm Ca ion), 2.0 μg/mL ofheat-treated extract total soluble protein containing an error-prone PCR(epPCR)-generated variant perhydrolase prepared as described in Example6) using 0 g/L or 2 g/L of liquid laundry detergent (comprising 15-30%anionic and non-ionic surfactants, 5-15% soap, and <5% polycarboxylates,perfume, phosphonates, optical brighteners) and compared to PAAproduction using the same concentration of C277T variant perhydrolase(Published U.S. Patent Application No. 2010-0087529 to DiCosimo et al.,SEQ ID NO: 5). The results of the various comparisons under simulatedlaundry care conditions are provided in Tables 6, 7 and 8. Under thesereaction conditions, variants 006B12 (W28C/F104S/C277T, SEQ ID NO: 16),006F12 (R261S/I264F/C277T, SEQ ID NO: 12), 006D10(W28C/L32P/D151E/C277T, SEQ ID NO: 14), 006E10 (R218C/C277T/F317L, SEQID NO: 34) and 006A10 (F268S/C277T, SEQ ID NO: 30) show a measurableimprovement in specific activity for peracetic acid production whencompared to the T. maritima C277T perhydrolase (SEQ ID NO: 5) under thespecified conditions (either with or without added liquid detergent atpH 10.0).

TABLE 6 Peracetic acid (PAA) production at 5 minutes and 20 minutes fromvariant 006B12 vs. T. maritima C277T in hard water (HW) (pH 10) at 20°C., 2.0 μg/mL heat-treated extract total soluble protein and 0 mg/mL or2.0 mg/mL liquid laundry detergent. Total soluble Liquid PAA @ PAA @Variant SEQ ID Triacetin H₂O₂ ¹ protein detergent 5 min. 20 min. ID NO:Samples (mM) (mM) (μg/mL) (mg/mL) Initial pH (ppm) (ppm) no perhydrolase2 10 0 0 10 16 45 Control 5 C277T 2 10 2.0 0 10 74 123 Control 5 C277T 210 2.0 0 10 67 106 006B12 16 W28C/F104S/C277T 2 10 2.0 0 10 79 123006B12 16 W28C/F104S/C277T 2 10 2.0 0 10 79 123 no perhydrolase 2 10 0 210 28 27 Control 5 C277T 2 10 2.0 2 10 51 — Control 5 C277T 2 10 2.0 210 50 89 006B12 16 W28C/F104S/C277T 2 10 2.0 2 10 64 96 006B12 16W28C/F104S/C277T 2 10 2.0 2 10 59 86 ¹= Hydrogen peroxide generated fromsodium percarbonate.

TABLE 7 Peracetic acid (PAA) production at 5 minutes and 20 minutes fromvariants 006F12 and 006D10 vs. T. maritima C277T in hard water (HW) andcarbonate buffer (pH 10) at 20° C., 2.0 μg/mL heat-treated extract totalsoluble protein and 0 mg/mL or 2.0 mg/mL liquid laundry detergent. Totalsoluble Liquid PAA @ PAA @ Variant SEQ ID Triacetin H₂O₂ ¹ proteindetergent 5 min. 20 min. ID NO: Samples (mM) (mM) (μg/mL) (mg/mL)Initial pH (ppm) (ppm) no perhydrolase 2 10 0 0 10 16 45 Control 5 C277T2 10 2.0 0 10 55 112 Control 5 C277T 2 10 2.0 0 10 55 113 006F12 12R261S/I264F/C277T 2 10 2.0 0 10 56 89 006F12 12 R261S/I264F/C277T 2 102.0 0 10 56 94 006D10 14 W28C/L32P/D151E/C277T 2 10 2.0 0 10 53 87006D10 14 W28C/L32P/D151E/C277T 2 10 2.0 0 10 54 92 no perhydrolase 2 100 2 10 28 27 Control 5 C277T 2 10 2.0 2 10 40 79 Control 5 C277T 2 102.0 2 10 39 75 006F12 12 R261S/I264F/C277T 2 10 2.0 2 10 56 89 006F12 12R261S/I264F/C277T 2 10 2.0 2 10 56 91 006D10 14 W28C/L32P/D151E/C277T 210 2.0 2 10 47 84 006D10 14 W28C/L32P/D151E/C277T 2 10 2.0 2 10 48 83 ¹=Hydrogen peroxide generated from sodium percarbonate.

TABLE 8 Peracetic acid (PAA) production at 5 minutes and 20 minutes fromvariants 006E10 and 006A10 vs. T. maritima C277T in hard water (HW) (pH10) at 20° C., 2.0 μg/mL heat-treated extract total soluble protein and0 mg/mL liquid laundry detergent. Total soluble Liquid PAA @ PAA @Variant SEQ Triacetin H₂O₂ ¹ protein detergent Initial 5 min. 20 min. IDID NO: Samples (mM) (mM) (μg/mL) (mg/mL) pH (ppm) (ppm) no perhydrolase2 10 0 0 10 23 57 Control 5 C277T 2 10 2.0 0 10 64 121 Control 5 C277T 210 2.0 0 10 64 120 006E10 34 R218C/C277T/F317L 2 10 2.0 0 10 73 125006E10 34 R218C/C277T/F317L 2 10 2.0 0 10 73 125 006A10 30 F268S/C277T 210 2.0 0 10 78 126 006A10 30 F268S/C277T 2 10 2.0 0 10 76 126 ¹=Hydrogen peroxide generated from sodium percarbonate.

Example 9 Comparison of Thermotoga maritima Perhydrolase Variants Vs.Thermotoga maritima C277T Perhydrolase Under Simulated Laundry CareApplication Conditions

Two error-prone PCR (epPCR)-generated variant perhydrolases were assayedfor peracetic acid (PAA) production under laundry application conditions(10 mL total volume, 1 mM triacetin (TA), 4.4 mM H₂O₂ from sodiumpercarbonate in hard water (HW, 400 ppm Ca ion), 1.0 μg/mL ofheat-treated extract total soluble protein containing an error-prone PCR(epPCR)-generated variant perhydrolase prepared as described in Example6) without added liquid laundry detergent and compared to PAA productionusing the same concentration of C277T variant perhydrolase (PublishedU.S. Patent Application No. 2010-0087529 to DiCosimo et al., SEQ ID NO:5) (Table 9). Under these reaction conditions, variants 006G11(D254G/C277T, SEQ ID NO: 46) and 006E12 (H227L/T233A/C277T/A290V, SEQ IDNO: 40) show a measurable improvement specific activity for peraceticacid production when compared to the C277T perhydrolase (SEQ ID NO: 5)from which the variants were prepared.

TABLE 9 Peracetic acid (PAA) production at 5 minutes and 20 minutes fromvariants 006G11 and 006E12 vs. T. maritima C277T in hard water (HW) (pH9) at 20° C., 1.0 μg/mL heat-treated extract total soluble protein and 0mg/mL liquid laundry detergent. Total soluble Liquid PAA @ PAA @ VariantSEQ ID Triacetin H₂O₂ ¹ protein detergent 5 min. 20 min. ID NO: Samples(mM) (mM) (μg/mL) (mg/mL) Initial pH (ppm) (ppm) no perhydrolase 1 4.4 00 9 1.6 6.5 Control 5 C277T 1 4.4 1 0 9 9.0 26 Control 5 C277T 1 4.4 1 09 9.3 26 006G11 46 D254G/C277T 1 4.4 1 0 9 13 33 006G11 46 D254G/C277T 14.4 1 0 9 13 32 006E12 40 H227L/T233A/C277T/A290V 1 4.4 1 0 9 12 31006E12 40 H227L/T233A/C277T/A290V 1 4.4 1 0 9 12 31 ¹= Hydrogen peroxidegenerated from sodium percarbonate.

Example 10 Comparison of Perhydrolase Variants Vs. Thermotoga maritimaC277S Variant Perhydrolase Using 2 mM 4-Acetoxybenzoic Acid and 10 mMH₂O₂ in Sodium Bicarbonate Buffer (10 mM, pH 6.5) at 20° C.

Reactions (100 mL total volume) were run at 20° C. in sodium bicarbonatebuffer (10 mM, pH 7.0) containing 4-acetoxybenzoic acid (4ABA, 2 mM),hydrogen peroxide (10 mM) and 2.0 μg/mL of heat-treated extract totalsoluble protein containing an error-prone PCR (epPCR)-generated variantperhydrolase prepared as described in Example 6. Reactions were stirredfor only the first 45 seconds of reaction to initially mix the reactantsand enzyme. A comparative reaction was also run under identicalconditions to that described immediately above using 2.0 μg/mL ofheat-treated extract total soluble protein isolated from E. coliKLP18/pSW228/C277S (expressing Thermotoga maritima C277S perhydrolasevariant, SEQ ID NO: 69), where the heat-treated extract supernatant wasprepared according to the procedure of Example 6, and where thisheat-treated extract supernatant contained the same concentration ofThermotoga maritima C277S perhydrolase variant (SEQ ID NO: 69) as apercentage of total soluble protein as was present in the heat-treatedextract total soluble protein containing an error-prone PCR(epPCR)-generated variant perhydrolase. Variant 873B4 (A266P/C277S, SEQID NO: 102) showed a measurable improvement in specific activity forperacetic acid production at 5 min when compared to the C277Sperhydrolase (SEQ ID NO: 69) from which the variant was prepared (Table10).

TABLE 10 Peracetic acid (PAA) production at 5 minutes and 20 minutesfrom variants 873A4 and 873B4 vs. T. maritima C277S in sodiumbicarbonate buffer (pH 7.0) at 20° C. with 2.0 μg/mL heat-treatedextract total soluble protein. Total soluble PAA @ PAA @ 4ABA H₂O₂protein Initial 5 min. 20 min. Variant ID SEQ ID NO: Samples (mM) (mM)(μg/mL) pH (ppm) (ppm) no perhydrolase 2 10 0 7.0 6 45 Control 69 C277S2 10 2.0 7.0 15 48 Control 69 C277S 2 10 2.0 7.0 18 50 873A4 98L125M/C277S 2 10 2.0 7.0 13 43 873A4 98 L125M/C277S 2 10 2.0 7.0 16 49873B4 102 A266P/C277S 2 10 2.0 7.0 23 56 873B4 102 A266P/C277S 2 10 2.07.0 21 52

Example 11 Comparison of a Thermotoga maritima Perhydrolase Variant Vs.Thermotoga maritima C277T Perhydrolase Under Simulated Laundry CareApplication Conditions

An error-prone PCR (epPCR)-generated variant perhydrolase was evaluatedfor peracetic acid (PAA) production under laundry application conditions(10 mL total volume, 0.5 mM triacetin (TA), 2.0 mM H₂O₂ from sodiumpercarbonate at 25° C. in deionized water, 2.0 μg/mL of heat-treatedextract total soluble protein containing an error-prone PCR(epPCR)-generated variant perhydrolase prepared as described in Example6) using 2 g/L of solid laundry detergent (a formulation generallycomprising surfactants (sodium dodecylbenzenesulfonate, C12-15 Pareth-5,C12-15 Pareth-7, sodium stearate, and stearic acid), builders (sodiumcarbonate, zeolite, sodium silicate, and citric acid), binders(cellulose, PEG-75, dextrin, and sucrose), bulking agents (sodiumsulfate, sodium chloride, sodium bicarbonate, and calcium carbonate),structurants (sodium acrylic Acid/MA copolymer, and sodiumpolyacrylate), sequestrants (tetrasodium etidronate and calcium sodiumEDTMP), optical brighteners (disodiumanilinomorpholinotriazinylaminostilbenesulfonate), stabilizing agents,anti-redeposition agents, antifoaming agents, and softness extenders)and compared to PAA production using the same concentration of C277Tvariant perhydrolase (Published U.S. Patent Application No. 2010-0087529to DiCosimo et al., SEQ ID NO: 5). The results of the variouscomparisons under simulated laundry care conditions are provided inTable 11. Under these reaction conditions, variant 006E10(R218C/C277T/F317L, SEQ ID NO: 34) showed a measurable improvement inspecific activity for peracetic acid production when compared to the T.maritima C277T perhydrolase (SEQ ID NO: 5) under the specifiedconditions (with added solid detergent at pH 10.7).

TABLE 11 Peracetic acid (PAA) production at 5 minutes and 20 minutesfrom variant 006E10 vs. T. maritima C277T in 2 mg/mL solid laundrydetergent (pH 10.7) at 25° C., 2.0 μg/mL heat-treated extract totalsoluble protein. Total soluble Solid PAA @ PAA @ PAA @ Variant SEQTriacetin H₂O₂ ¹ protein detergent Initial 5 min. 10 min 20 min. ID IDNO: Samples (mM) (mM) (μg/mL) (mg/mL) pH (ppm) (ppm) (ppm) noperhydrolase 0.5 2.0 0 2 10.7 2.2 4.3 5.6 Control 5 C277T 0.5 2.0 2.0 210.7 7.5 11 13 006E10 34 R218C/C277T/F317L 0.5 2.0 2.0 2 10.7 11 14 15¹= Hydrogen peroxide generated from sodium percarbonate.

1. An isolated nucleic acid molecule selected from the group consistingof: (a) a polynucleotide encoding a polypeptide having perhydrolyticactivity, said polypeptide comprising the amino acid sequence of SEQ IDNO: 34; (b) a polynucleotide comprising the nucleic acid sequence of SEQID NO: 33; and (c) a polynucleotide fully complementary to thepolynucleotide of (a) or (b).
 2. A vector comprising the nucleic acidmolecule of claim
 1. 3. A recombinant DNA construct comprising thenucleic acid molecule of claim 1 operably-linked to a suitableregulatory sequence.
 4. A recombinant host cell comprising therecombinant DNA construct of claim
 3. 5. A method for expressing apolypeptide comprising transforming a host cell with the recombinant DNAconstruct of claim
 3. 6. An isolated polypeptide having perhydrolyticactivity comprising the amino acid sequence of SEQ ID NO:
 34. 7. Thepolypeptide of claim 6; wherein said polypeptide is characterized by aperacetic acid formation specific activity that is higher than theperacetic acid formation specific activity of the Thermotoga maritimaC277T acetyl xylan esterase having amino acid sequence SEQ ID NO:
 5. 8.A process for producing a peroxycarboxylic acid comprising: (a)providing a set of reaction components comprising: (1) at least onesubstrate selected from the group consisting of: (i) one or more estershaving the structure[X]_(m)R₅ wherein X=an ester group of the formula R₆—C(O)O; R₆═C1 to C7linear, branched or cyclic hydrocarbyl moiety, optionally substitutedwith hydroxyl groups or C1 to C4 alkoxy groups, wherein R₆ optionallycomprises one or more ether linkages for R₆═C2 to C7; R₅=a C1 to C6linear, branched, or cyclic hydrocarbyl moiety or a five-membered cyclicheteroaromatic moiety or six-membered cyclic aromatic or heteroaromaticmoiety optionally substituted with hydroxyl groups; wherein each carbonatom in R₅ individually comprises no more than one hydroxyl group or nomore than one ester group or carboxylic acid group; wherein R₅optionally comprises one or more ether linkages; m is an integer rangingfrom 1 to the number of carbon atoms in R₅; and wherein said esters havesolubility in water of at least 5 ppm at 25° C.; (ii) one or moreglycerides having the structure

wherein R₁═C1 to C21 straight chain or branched chain alkyl optionallysubstituted with an hydroxyl or a C1 to C4 alkoxy group and R₃ and R₄are individually H or R₁C(O); (iii) one or more esters of the formula:

wherein R₁ is a C1 to C7 straight chain or branched chain alkyloptionally substituted with an hydroxyl or a C1 to C4 alkoxy group andR₂ is a C1 to C10 straight chain or branched chain alkyl, alkenyl,alkynyl, aryl, alkylaryl, alkylheteroaryl, heteroaryl, (CH₂CH₂O)_(n), or(CH₂CH(CH₃)—O)_(n)H and n is 1 to 10; (iv) one or more acylatedmonosaccharides, acylated disaccharides, or acylated polysaccharides;and (v) any combination of (i) through (iv); (2) a source of peroxygen;and (3) an enzyme catalyst comprising the polypeptide of claim 6; (b)combining the set of reaction components under suitable reactionconditions whereby peroxycarboxylic acid is produced; and (c) optionallydiluting the peroxycarboxylic acid produced in step (b).
 9. The processof claim 8 further comprising the step of: d) contacting a hard surfaceor inanimate object with the peroxycarboxylic acid produced in step (b)or step (c); whereby said hard surface or said inanimate object isdisinfected, bleached, destained or a combination thereof.
 10. Theprocess of claim 8 wherein the inanimate object is a medical instrument.11. The process of claim 8 further comprising the step of: d) contactingan article of clothing or a textile with peroxycarboxylic acid producedin step (b) or step (c); whereby the article of clothing or textilereceives a benefit.
 12. The process of claim 11 wherein the benefit isselected from the group consisting of a disinfecting, sanitizing,bleaching, destaining, deodorizing, and combinations thereof.
 13. Theprocess of claim 8 further comprising the step of: d) contacting woodpulp or paper pulp with peroxycarboxylic acid produced in step (b) orstep (c); whereby the wood pulp or paper pulp is bleached.
 14. Theprocess of claim 8 wherein the substrate is selected from the groupconsisting of: monoacetin; diacetin; triacetin; monopropionin;dipropionin; tripropionin; monobutyrin; dibutyrin; tributyrin; glucosepentaacetate; 13-D-galactose pentaacetate, sorbitol hexaacetate, sucroseoctaacetate, xylose tetraacetate; acetylated xylan; acetylated xylanfragments; 13-D-ribofuranose-1,2,3,5-tetraacetate;tri-O-acetyl-D-galactal; tri-O-acetyl-D-glucal; monoesters or diestersof 1,2-ethanediol, 1,2-propanediol, 1,3-propanediol, 1,2-butanediol,1,3-butanediol, 2,3-butanediol, 1,4-butanediol, 1,2-pentanediol,2,5-pentanediol, 1,6-pentanediol, 1,2-hexanediol, 2,5-hexanediol,1,6-hexanediol; 4-acetoxybenzoic acid; and mixtures thereof.
 15. Theprocess of claim 14 wherein the substrate is triacetin.
 16. The processof claim 8 wherein the peroxycarboxylic acid produced is peracetic acid,perpropionic acid, perbutyric acid, perlactic acid, perglycolic acid,permethoxyacetic acid, per-β-hydroxybutyric acid, or mixtures thereof.17. The process of claim 8 wherein the enzyme catalyst is in the form ofa microbial cell, a permeabilized microbial cell, a microbial cellextract, a partially purified enzyme, or a purified enzyme.
 18. Acomposition comprising: (a) a set of reaction components comprising: (1)at least one substrate selected from the group consisting of: (i) one ormore esters having the structure[X]_(m)R₅ wherein X=an ester group of the formula R₆—C(O)O; R₆═C1 to C7linear, branched or cyclic hydrocarbyl moiety, optionally substitutedwith hydroxyl groups or C1 to C4 alkoxy groups, wherein R₆ optionallycomprises one or more ether linkages for R₆═C2 to C7; R₅=a C1 to C6linear, branched, or cyclic hydrocarbyl moiety or a five-membered cyclicheteroaromatic moiety or six-membered cyclic aromatic or heteroaromaticmoiety optionally substituted with hydroxyl groups; wherein each carbonatom in R₅ individually comprises no more than one hydroxyl group or nomore than one ester group or carboxylic acid group; wherein R₅optionally comprises one or more ether linkages; m is an integer rangingfrom 1 to the number of carbon atoms in R₅; and wherein said esters havesolubility in water of at least 5 ppm at 25° C.; (ii) one or moreglycerides having the structure

wherein R₁═C1 to C21 straight chain or branched chain alkyl optionallysubstituted with an hydroxyl or a C1 to C4 alkoxy group and R₃ and R₄are individually H or R₁C(O); (iii) one or more esters of the formula:

wherein R₁ is a C1 to C7 straight chain or branched chain alkyloptionally substituted with an hydroxyl or a C1 to C4 alkoxy group andR₂ is a C1 to C10 straight chain or branched chain alkyl, alkenyl,alkynyl, aryl, alkylaryl, alkylheteroaryl, heteroaryl, (CH₂CH₂O)_(n), or(CH₂CH(CH₃)—O)_(n)H and n is 1 to 10; (iv) one or more acylatedmonosaccharides, acylated disaccharides, or acylated polysaccharides;and (v) any combination of (i) through (iv); (2) a source of peroxygen;and (3) an enzyme catalyst comprising the polypeptide of claim 6; and(b) at least one peroxycarboxylic acid formed upon combining the set ofreaction components of (a).
 19. A peracid generation and delivery systemcomprising: (a) a first compartment comprising (1) an enzyme catalystcomprising the polypeptide of claim 6; (2) at least one substrateselected from the group consisting of: (i) one or more esters having thestructure[X]_(m)R₅ wherein X=an ester group of the formula R₆—C(O)O; R₆═C1 to C7linear, branched or cyclic hydrocarbyl moiety, optionally substitutedwith hydroxyl groups or C1 to C4 alkoxy groups, wherein R₆ optionallycomprises one or more ether linkages for R₆═C2 to C7; R₅=a C1 to C6linear, branched, or cyclic hydrocarbyl moiety or a five-membered cyclicheteroaromatic moiety or six-membered cyclic aromatic or heteroaromaticmoiety optionally substituted with hydroxyl groups; wherein each carbonatom in R₅ individually comprises no more than one hydroxyl group or nomore than one ester group or carboxylic acid group; wherein R₅optionally comprises one or more ether linkages; m is an integer rangingfrom 1 to the number of carbon atoms in R₅; and wherein said esters havesolubility in water of at least 5 ppm at 25° C.; (ii) one or moreglycerides having the structure

wherein R₁═C1 to C21 straight chain or branched chain alkyl optionallysubstituted with an hydroxyl or a C1 to C4 alkoxy group and R₃ and R₄are individually H or R₁C(O); (iii) one or more esters of the formula:

wherein R₁ is a C1 to C7 straight chain or branched chain alkyloptionally substituted with an hydroxyl or a C1 to C4 alkoxy group andR₂ is a C1 to C10 straight chain or branched chain alkyl, alkenyl,alkynyl, aryl, alkylaryl, alkylheteroaryl, heteroaryl, (CH₂CH₂O)_(n), or(CH₂CH(CH₃)—O)_(n)H and n is 1 to 10; (iv) one or more acylatedmonosaccharides, acylated disaccharides, or acylated polysaccharides;and (v) any combination of (i) through (iv); and (3) an optional buffer;and (b) a second compartment comprising (1) source of peroxygen; (2) aperoxide stabilizer; and (3) an optional buffer.
 20. The peracidgeneration and delivery system of claim 19 wherein the substratecomprises triacetin.
 21. A laundry care product comprising thepolypeptide of claim
 6. 22. A personal care product comprising thepolypeptide of claim 6.